Monoclonal antibody capable of binding integrin alpha 10 beta 1

ABSTRACT

The present invention provides a monoclonal antibody or a fragment thereof binding to the extracellular I-domain of integrin alpha10beta1 and a hybridoma cell line deposited at the Deutsche Sammlung von Microorganismen und Zellkulturen GmbH under the accession number DSM ACC2583. Furthermore, the present invention also provides a monoclonal antibody or a fragment thereof binding to the extracellular I-domain of integrin alpha10beta1 produced by the hybridoma cell line deposited. Methods and uses of said antibody or a fragment thereof in identifying and selecting cells of a chondrogenic nature for treatment purposes, in particular for the identification and isolation of chondrocytes, mesenchymal progenitor cells and embryonic stem cells for tissue engineering of cartilage, or for identifying diagnostic and therapeutic tools in studying the biological role and the structural/functional relationships of the integrin alpha10beta1 with its various extracellular matrix ligands are also included.

This application is a continuation of U.S. application Ser. No.10/553,226, filed Feb. 6, 2006, which is the National Stage ofInternational Application No. PCT/SE2004/000580, filed Apr. 14, 2004,which claims the benefit of priority of Swedish Application No.0301087-3, filed Apr. 14, 2003, and U.S. Provisional Application No.60/320,112, filed Apr. 16, 2003. All of the above applications areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The invention relates to the generation of novel monoclonal antibodiesor fragments thereof to the I-domain of the integrin alpha10 chain(α10), and a hybridoma cell-line expressing one such antibody as well asmethods for using antibodies or fragments thereof for diagnostic,analytical and therapeutic purposes.

BACKGROUND OF THE INVENTION Integrins

Integrins are glycoprotein heterodimers that contain a covalentlyassociated alpha and beta subunit. The integrin subunits aretransmembrane proteins possessing an extracellular domain forinteracting with an extracellular matrix or cellular component, atransmembrane domain spanning the cell membrane, and a cytoplasmicdomain for interacting with one or more skeletal components. To date,there are eighteen known alpha subunits that can combine with eightknown beta subunits (Gullberg and Lundgren-Åkerlund, 2002), resulting inat least twenty-four different integrin molecules. Integrins can begrouped into subfamilies depending on which beta subunit they contain oralternatively the grouping can be based upon shared structural featuresof the alpha chain i.e. those integrins characterised by the presence ofan additional region known as the I (inserted)-domain. This groupincludes nine members and thus represents half of the currently knownintegrin alpha chains (Velling 1999).

Integrin Alpha10Beta1

Recently we discovered a new collagen-binding integrin heterodimer(Camper et al 1998) that contains a novel alpha chain, designatedalpha10. This alpha chain is associated with a beta1 subunit(alpha10beta1) and is a member of the I-domain containing integrins.Currently 4 collagen-binding I-domain containing integrins are known,alpha1beta 1, alpha2beta1, alpha10beta1 and alpha11beta1 (Gullberg andLundgren-Åkerlund 2002).

Sequence analysis shows that alpha10 has the highest identity withalpha11 (43%) and an identity of 33% with alpha1 and 31% with alpha2.

Expression of Integrin Alpha10Beta1

Integrin alpha10beta1 is mainly expressed on chondrocytes in articularcartilage, in the vertebral column, in trachea and in the cartilagesupporting the bronchi (Camper et al 2001). The integrin is also foundin specialized fibrous tissues such as the fascia of skeletal muscle andtendon, in the ossification groove of Ranvier and in the aortic andatrioventricular valves of the heart (Camper et al 2001).

Function of Integrin Alpha10Beta1 in Cartilage

Chondrocytes are the only cell type in articular cartilage and areresponsible for the coordinated synthesis and turnover of theextracellular matrix (ECM) components of the tissue. The two maincomponents of the ECM, apart from water, are different types of collagenand the large aggregating proteoglycan aggrecan. The integrinalpha10beta1 on the chondrocyte cell surface mediates the binding ofcollagen to the chondrocyte and, like other integrin-extracellularmatrix protein interactions (Heino 2000, Boudreau and Jones 1999, Hering1999), is likely to be responsible for signalling the dynamic state ofthe surrounding matrix to the cell. Although collagen type II is likelyto be an important ligand for alpha10beta1, it is not a prerequisite foralpha10beta1 expression since alpha10beta1 is also present in tissuesthat lack collagen type II. This implies that alpha10beta1 in vivo canbind to other important extracellular matrix ligands such aschondroadherin and other collagen types e.g. type I and type VI (Tullaet al. 2001).

Identifying tools for studying the biological role and thestructural/functional relationships of this integrin with its variousextracellular matrix ligands is therefore of great value. Such tools maybe of a diagnostic nature for the detection of the presence ofalpha10beta1, or maybe of therapeutic value in blocking or stimulatingthe activity of alpha10beta1.

Antibodies to Integrin Alpha10

Camper et al. (1998) describes the generation of polyclonal antibodiesto the cytoplasmic domain of integrin alpha10beta1. The cytoplasmicdomain consists of a 16 amino acid (Armulik 2000) sequence extendingfrom the transmembrane domain. This domain is therefore is an idealimmunogen and production of polyclonal antibodies to this domain byimmunisation with a peptide, whose sequence corresponds to a regionwithin the cytoplasmic domain, is therefore a relatively simple,straightforward procedure routinely carried out to produce antibodies.(Harlow and Lane 1988). The polyclonal antibodies of Camper et al.(1998) generated in rabbit are of limited use since they are unable tobe used on living cells due to their inability to penetrate cells.

General Structure of Naturally Occurring Antibodies

Naturally occurring antibodies comprise of two heavy chains linkedtogether by disulphide bonds and two light chains, one light chain beinglinked to each heavy chain by disulphide bonds. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain (V_(L)) at one end and aconstant domain at its other end.

It is the variable domains of each pair of light and heavy chains thatare directly involved in binding the antibody to the antigen (Harlow andLane (1999)). The domains of the natural light and heavy chains have thesame general structure and each domain comprises of four framework (Fr)regions, whose sequences are somewhat conserved, connected by threehyper-variable or complementarity determining regions (CDRs).

Monoclonal Antibodies of Non-Human Origin in Therapeutic Applications

Murine-derived monoclonal antibodies may cause an immunogenic responsein human patients, reducing their therapeutic applicability. Tocircumvent this problem, humanised antibodies have therefore beendeveloped in which the murine antigen binding variable domain is coupledto a human constant domain. (Morrison et al (1984), Boulianne et al(1984), Neuberger et al (1985)).

In a further effort to resolve antigen-binding functions of antibodiesand to minimise the use of heterologous sequences in human antibodies,the CDRs or CDR sequences of murine antibodies are grafted onto thehuman variable region framework (Jones et al 1986, Riechmann et al 1988,Verhoeyen et al 1988). The therapeutic efficacy of this approach hasbeen demonstrated previously (Reichmann et al (1988) and Hale et al(1989)).

Monoclonal Antibodies in Joint Diseases

Mature articular cartilage has no blood vessels, it is not innervatedand normal mechanisms of tissue repair, involving the recruitment ofcells to the site of damage does not occur. This means that cartilagehas a very poor reparative response to injury and its irreparablebreakdown is a common feature of degenerative joint diseases. Repair ofsuch injuries has focused upon different tissue engineering strategiesthat involve the delivery or in situ mobilisation of cells capable ofrestoring the pathologically altered architecture and function of thetissue. Tissue engineering approaches for cartilage currently useisolated autologous cells derived from biopsies from healthy siteswithin the cartilage (autologous chondrocyte transplantation—ACT)(Brittberg 1999). Critical to ACT is the quality of the cells that areimplanted back into the joint i.e. the cells should be chondrocytescapable of producing a hyaline-like cartilage (Jobanputra et al 2001).

An alternative strategy to the use of autologous chondrocytes is the useof stem cells with a chondrogenic differentiation capacity such asmesenchymal stem cells (FIG. 1) that can be used in vivo to repair orgenerate new cartilage (Jorgensen et al 2001, Johnstone and Yoo 2001).Whilst it is well documented that MSCs have the inherent potential todifferentiate into osteogenic, chondrogenic, adipogenic and myocardiaccell lineages, there is currently no means of identifying the progenitorcell that will lead to these different lineages. Markers exist toindicate whether the cell is capable of expressing a cartilage phenotypei.e. collagen II and aggrecan, but these proteins are expressedextracellularly after synthesis, and cannot be used for isolation of achondrogenic cell type.

Antibodies against extracellular integrin epitopes, in contrast tointracellular integrin epitopes, are in general difficult to generatedue to a low or absent immunogenic capacity. Normally, this problem issolved by the skilled artisan by administering an adjuvant in parallelwith the antigen of interest. Different adjuvants exist and by using oneor another, or a combination thereof, a more or less general activationof the host's immune system is generated. Still, as of today's date andwith the known accumulated knowledge of adjuvants, no monoclonalantibodies against the extracellular parts of integrin alpha10beta1 havebeen generated. Thus, an antibody useful in therapy, diagnosis and insitu studies of joint diseases is currently lacking due to thedifficulty identified in generating such antibodies.

The one distinguishable feature common to the primary collagen bindingintegrins receptors is the existence of an I (“inserted”) domain at theN-terminal of the alpha subunit. Only four collagen-binding integrinsexist that contain an I-domain (integrin alpha1beta1, alpha2beta1,alpha10beta1 and alpha 11beta1). The I-domains still only show anoverall identity of maximum of 60%. The I-domain of the integrin alpha10is of particular interest since this domain contains unique structuraldifferences compared to the I-domains of the other collagen-bindingintegrins. These differences include the number of cysteine residues,the high degree of positive amino acids and the recognition of distinctcollagen subtypes (Gullberg and Lundgren-Åkerlund 2002, Tulla et al2001). The I-domain thus comprises a unique ligand binding part and itis thus highly desirable to generate monoclonal antibodies against theI-domain of integrin alpha10, and integrin alpha10beta1.

It is further highly desirable to provide a tool that could identify andselect cells of a chondrogenic nature for treatment purposes, inparticular for the isolation of chondrocytes, mesenchymal progenitorcells and embryonic stem cells for tissue engineering of cartilage.

It is further highly desirable in the light of aforementioned problemsto develop means and methods for identifying diagnostic and therapeutictools in studying the biological role and the structural/functionalrelationships of the integrin alpha10beta1 with its variousextracellular matrix ligands. Further, there is an unmet need toidentifying blocking or neutralizing and stimulatory agents,particularly for chondrocytes, mesenchymal stem cells and other cellsexpressing the integrin alpha10beta1. In this respect, the presentinvention addresses these needs and interest.

SUMMARY OF THE INVENTION

In view of the foregoing disadvantages known in the art when identifyingand selecting cells of a chondrogenic nature for treatment purposes, inparticular for the identification and isolation of chondrocytes,mesenchymal progenitor cells and embryonic stem cells for tissueengineering of cartilage, or for identifying diagnostic and therapeutictools in studying the biological role and the structural/functionalrelationships of the integrin alpha10beta1 with its variousextracellular matrix ligands, the present invention provides amonoclonal antibody or fragments thereof, specific for the I-domain ofthe integrin alpha10beta1, a cell line producing said monoclonalantibody and as well as methods and uses for different diseases relatedto joints, cartilage and atherosclerosis.

One object with the present invention is to provide a highly specificantibody for binding to the extracellular I-domain of integrinalpha10beta1.

Thus, the present invention provides a monoclonal antibody or a fragmentthereof binding to the extracellular I-domain of integrin alpha10beta1.

Also, the present invention provides a hybridoma cell line deposited atthe Deutsche Sammlung von Microorganismen und Zellkulturen GmbH underthe accession number DSM ACC2583.

Furthermore, the present invention also provides a monoclonal antibodyor a fragment thereof binding to the extracellular I-domain of integrinalpha10beta1 produced by the hybridoma cell line deposited at theDeutsche Sammlung von Microorganismen und Zellkulturen GmbH under theaccession number DSM ACC2583.

Still furthermore, the invention provides a method for isolating apopulation of mammalian mesenchymal stem cells. The method comprises thesteps of

-   -   a) providing a cell suspension comprising mammalian mesenchymal        stem cells,    -   b) contacting the cell suspension in a) with a monoclonal        antibody or a fragment thereof binding to the extracellular        I-domain of integrin alpha10beta1, under conditions wherein said        monoclonal antibody or a fragment thereof forms an        antibody-antigen complex with the extracellular domain of        integrin alpha10beta1,    -   c) separating cells binding to the monoclonal antibody or a        fragment thereof in b), and optionally    -   d) recovering cells binding to the monoclonal antibody or a        fragment thereof in c) from said antibody or a fragment thereof,        thereby producing a population of mammalian mesenchymal stem        cells, optionally free from said antibody or a fragment thereof.

Similarly, the invention provides a method for isolating a population ofmammalian chondrocytes. The method comprises the steps of

-   -   a) providing a cell suspension comprising chondrocytes,    -   b) contacting the cell suspension in a) with a monoclonal        antibody or a fragment thereof binding to the extracellular        domain of integrin alpha10beta1, under conditions wherein said        monoclonal antibody or a fragment thereof forms an        antibody-antigen complex with the extracellular I-domain of        integrin alpha10beta1,    -   c) separating cells binding to the monoclonal antibody or a        fragment thereof in b), and optionally    -   d) recovering cells binding to the monoclonal antibody or a        fragment thereof in c) from said antibody or a fragment thereof,        thereby producing a population of chondrocytes, optionally free        from said antibody or a fragment thereof.

Similarly, the invention provides a method for isolating asub-population of mammalian ES cells; the method comprises the steps of

-   -   a) providing a cell suspension comprising ES cells,    -   b) contacting the cell suspension in a) with a monoclonal        antibody or a fragment thereof binding to the extracellular        domain of integrin alpha10beta1, under conditions wherein said        monoclonal antibody or a fragment thereof forms an        antibody-antigen complex with the extracellular I-domain of        integrin alpha10beta1,    -   c) separating cells binding to the monoclonal antibody or a        fragment thereof in b), and optionally    -   d) recovering cells binding to the monoclonal antibody or a        fragment thereof in c) from said antibody or a fragment thereof,        thereby producing a sub-population of mammalian ES cells,        optionally free from said antibody or a fragment thereof.

Further embodiments of the methods above is wherein the monoclonalantibody or a fragment thereof binding to the extracellular domain ofintegrin alpha10beta1 is a monoclonal antibody or a fragment thereofbinding to the extracellular I-domain of integrin alpha10beta1 producedby the hybridoma cell line deposited at the Deutsche Sammlung vonMicroorganismen und Zellkulturen GmbH under the accession number DSMACC2583.

Further embodiments of the methods are wherein the monoclonal antibodyor a fragment thereof is linked to a solid phase.

The invention also provides a population of mammalian mesenchymal stemcells, a population of mammalian chondrocytes, and a sub-population ofmammalian embryonic stem cells obtainable by the methods describedabove.

The invention also provides uses of a monoclonal antibody or a fragmentthereof binding to the extracellular I-domain of integrin alpha10beta1,for the preparation of a pharmaceutical composition for the treatment ofa joint disease or atherosclerosis.

Further methods and uses are also provided and described in detailbelow.

SHORT DESCRIPTION OF DRAWINGS

FIG. 1 shows a schedule of lineage differentiation of totipotentembryonic stem (ES) cells to pluripotent adult stem cells capable offorming neural, haematopoietic, epithelial and mesenchymal stem cells(MSCs). Differentiation from ES cells to MSCs and further tochondrocytes shows the pathway of cells capable of expressing theintegrin alpha10beta1.

FIG. 2 shows immunoprecipitation using the antibody 365 and the I-domainof integrin alpha10beta1. The antibody 365 is able to immunoprecipitatethe whole integrin alpha10beta1 expressed on the surface of thealpha10-transfected C2C12 cells (lane 3); cytoplasmic polyclonal alpha10antibody was used as a positive control (lane 1) and cytoplasmicpolyclonal alpha11 antibody was used as a negative control (lane 2). Theantibody 365 was specific for the alpha10beta1 integrin since it did notimmunoprecipitate integrin alpha11beta1 from alpha11-transfected C2C12cells (lane 6). Polyclonal serum against the cytoplasmic domain ofintegrin alpha11 subunit (lane 5) was used a positive control andcytoplasmic polyclonal alpha10 antibody was used as a negative control(lane 4).

FIG. 3 shows a specificity test of the antibody 365 for alpha10 inELISA. No binding to alpha1 or alpha11 is observed. The absorbance ofthe colorimetric change was determined at 492 nm.

FIG. 4 shows results from a cell adhesion assay. mAb365 modulates thebinding of α10β1 integrin to type II collagen under defined conditions.a) mAb365 inhibits binding of α10β1-expressing C2C12 cells to collagenII in the presence of 1 mM Mg²⁺ and 1 mM Ca²⁺. Control (no Ab) and 1B4(isotype control) showed no inhibition of binding. b) Binding ofα11β1-expressing C2C12 cells to type II collagen is not inhibited bymAb365. Control (no Ab) and 1B4 (isotype control) showed no inhibitionof binding.

FIG. 5 shows identification of cells expressing alpha10 integrin byFACS-analysis. The antibody 365 bound to C2C12 cells transfected withhuman alpha10 integrin-subunit (upper middle panel). This was seen as adisplacement in the FACS histogram to the right. The antibody 365 didnot bind to C2C12 cells transfected with human alpha11 integrin-subunit(upper right panel) or untransfected C2C12 cells (upper left panel). Thelower panels represent secondary antibody alone, which did not bind toany of the cells tested.

FIG. 6 shows the results of positive selection by MACS® ofalpha10-expressing cells determined by flow cytometry analysis, FACS.Cells before selection, flow through and eluted cells were incubatedwith 365, and stained with PE labelled goat-anti-mouse IgG. Alpha10positive populations are shifted to the right as displayed in histograms5B.

FIG. 7 shows identification of a population of integrinalpha10-expressing hMNCs using the antibody 365 in MACS® analysis (lowerpanel). The upper panel shows MACS analysis in the absence of theantibody 365.

FIG. 8 shows detection of alpha10 in human articular cartilage using theantibody 365. Human articular cartilage sections were immunolocalisedwith the antibody 365 detected using a donkey anti-mouse secondaryantibody labelled with Cy3 (FIG. 8 a). Integrin alpha10beta1 expressionon human chondrocytes is clearly show clear of the when using theantibody 365. Control (secondary antibody only) does not bind to theintegrin alpha10beta1 (FIG. 8 b).

FIG. 9 shows percentage of alpha10⁺- and alpha10⁻-cells detected withmAb365 in FACS-analysis. Cells were analyzed on day 1, or after 1, 2,and 6 weeks.

FIG. 10 shows Collagen type II and Collagen type I mRNA levels in humanchondrocytes, separated upon the basis of their expression of alpha10,i.e. alpha10+ve or alpha10−ve, using mAb365 and magnetic cell sorting.

FIG. 11 shows that Mab365 recognises the alpha10 integrin expression onmouse chondrocytes. The histogram demonstrates the level of alpha10 andbeta1 integrin on mouse chondrocytes. Dotted line represents the cellsstained with isotype control antibody, filled histogram the alpha10stained with mAb365 and the solid line illustrates the beta1.

FIG. 12 shows the alpha10 integrin expression on human MSC. Thehistogram shows the level of alpha10 and beta 1 on hMSC. Dotted linerepresents the cells stained with isotype control antibody, filledhistogram the cells stained with mAb365 expressing alpha10, and thesolid line illustrates the integrin beta1 subunit.

FIG. 13 shows that Collagen type II mRNA levels are stimulated in thepresence of mAb365. Samples represent Control (no antibody), mAb365, andisotype control (IgG2akappa).

FIG. 14 shows the results after staining normal human articularcartilage by immunohistochemistry using the mAb365 antibody. Integrinalpha10beta1 can be detected in the tissue, here represented by twodifferent human specimens of 19 years and 53 years of age.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “Mesenchymal stem cells” refers to cells thatcan differentiate into a variety of differentiated cell types, includingcells forming bone, cartilage, muscle, tendons and ligaments, adiposetissue, and connective tissues.

As used herein, the term “Chondrocyte” refers to cells that comprisecartilage.

As used herein, the term “Chondrogenic” refers to those cells that havethe potential to become chondrocytes.

As used herein, “pharmaceutical composition” means therapeuticallyeffective composition according to the invention.

A “therapeutically effective amount”, or “effective amount”, or“therapeutically effective”, as used herein, refers to that amount whichprovides a therapeutic effect for a given condition and administrationregimen. This is a predetermined quantity of active material calculatedto produce a desired therapeutic effect in association with the requiredadditive and diluent; i.e., a carrier, or administration vehicle.Further, it is intended to mean an amount sufficient to reduce and mostpreferably prevent, a clinically significant deficit in the activity,function and response of the host. Alternatively, a therapeuticallyeffective amount is sufficient to cause an improvement in a clinicallysignificant condition in a host. As is appreciated by those skilled inthe art, the amount of a compound may vary depending on its specificactivity. Suitable dosage amounts may contain a predetermined quantityof active composition calculated to produce the desired therapeuticeffect in association with the required diluent; i.e., carrier, oradditive. In the methods and use for manufacture of compositions of theinvention, a therapeutically effective amount of the active component isprovided. A therapeutically effective amount can be determined by theordinary skilled medical or veterinary worker based on patientcharacteristics, such as age, weight, sex, condition, complications,other diseases, etc., as is well known in the art.

As used herein, the term “to modulate” is intended to mean a capacity toaffect a cell signalling effect directly or indirectly. To modulate thusmeans to act as an antagonist, i.e. partially or fully inhibit, reduce,alleviate, block or prevent; or to increase or stimulate, i.e. to act asan agonist. The modulation may be direct or indirect. By “indirectmodulation” the effect is not via a natural ligand binding site but viaanother site on the same molecule or via another second molecule. Thisis in contrast to “direct modulation” acting via a natural ligandbinding site.

A Hybridoma Cell Line

As revealed above, the present invention relates to antibodies, andhybridomas producing such antibodies, specific for the extracellularligand-binding domains of integrin alpha10beta1.

More specifically the present invention relates to one generatedhybridoma cell line producing an antibody specific for the extracellularligand-binding I-domain of integrin alpha10beta1. Thus, a hybridoma cellline deposited at the Deutsche Sammlung von Microorganismen undZellkulturen GmbH under the accession number DSM ACC2583 is disclosed.

A monoclonal antibody (mAb) or fragments thereof with specificity forthe extracellular ligand-binding I-domain of alpha10beta1 is of greatvalue in understanding the development, function, signalling, anddifferentiation of cells expressing this integrin, particularlymesenchymal stem cells, cells of a chondrogenic nature, chondrocytes,fibroblasts, tenocytes, myoblasts, osteoblasts, monocytes ormacrophages.

FIG. 1 shows a schedule of lineage differentiation of totipotentembryonic stem (ES) cells to pluripotent adult stem cells capable offorming neural, haematopoietic, epithelial and mesenchymal stem cells(MSCs). Differentiation from ES cells to MSCs and further tochondrocytes shows the pathway of cells capable of expressing theintegrin alpha10beta1.

Accordingly, the antibodies specific to the I-domain of the integrinsubunit alpha10, such as the antibody 365 produced by the hybridoma cellline mAb 365 with deposit number DSM ACC2583, of the present inventionmay also be used to modulate receptor function in research andtherapeutic applications. For instance, the antibodies described hereinmay act as antagonist to inhibit, i.e. reduce or prevent, or act asagonist, i.e. increase or stimulate, (a) binding e.g., of a ligand tothe receptor, (b) a receptor signalling function, and/or (c) astimulatory function. Antibodies that may act as agonists or antagonistsof receptor function may block ligand binding directly or indirectlye.g., by causing a conformational change. For example, antibodies mayinhibit receptor function by inhibiting binding of a ligand, or bydesensitization, with or without inhibition of binding of a ligand.Antibodies which bind receptor may also act as agonists of receptorfunction, triggering or stimulating a receptor function, such as asignalling and/or a stimulatory function of a receptor e.g., modulatingextracellular matrix (ECM) turnover, stimulating ECM synthesis.

Even more importantly, a modulatory, e.g. stimulatory, blocking orinhibitory, mAb that binds to cells expressing the integrin alpha10beta1have a great potential as a therapeutic agent.

Furthermore, a mAb specific to the I-domain of integrin alpha10beta1 maybe used as a drug delivery vehicle, or in combination with known drugdelivery vehicles.

Furthermore, a mAb specific to the I-domain of integrin alpha10beta1 maybe used to target the cell surface of cells expressing the integrinalpha10beta1 in gene therapy.

Generation of a mAb Specific for the I-Domain of the Integrin Alpha10Subunit

Due to problems in generating monoclonal antibodies specific for theI-domain of integrin alpha10 subunit, a specific protocol for generatingmonoclonal antibodies has been generated and evaluated. The protocol isexemplified below by generation of a cell line mAb 365 producing theantibody 365.

For the generation of the hybridoma cell line mAb 365, producing anantibody binding to the extracellular alpha10beta1 I-domain, a geneknockout mouse of the integrin alpha10beta1 was used. The knockout mouseis described in SE Application no 0201130-2 filed on 12 Apr. 2002,included herein by reference.

After immunisation and boosting, spleen cells were fused with NSO cellsand the resulting hybridoma cells cloned. Clone mAb 365 secreted amonoclonal antibody, 365, with specificity for alpha10beta1. As far asspecificity is concerned, the monoclonal antibody binds to alpha10beta1of both human and murine origin.

Example 1 gives a more detailed description of the generation of thecell line mAb 365.

According to the invention, a monoclonal antibody or fragments thereofagainst an extracellular region of the integrin alpha10beta1 produced bythe hybridoma cell line mAb 365 described above with the accessionnumber DSM ACC2583 is disclosed.

Monoclonal Antibody 365

The integrin alpha10beta1 is one of a member of 4 collagen bindingI-domain containing integrins. Like the other I-domain-containingcollagen binding integrins, the α10 I-domain contains a so-called MIDAS(metal ion-dependent adhesion site) motif. This motif is believed tohave an important role in ligand binding to the I-domain. Upon ligandbinding the conformation of the I-domain is altered and extensivechanges occur in the secondary and tertiary structure of the domain(Emsley et al 2000).

Molecular modelling of the α10 I-domain, based on the α2 I-domaincrystal structure, has revealed a higher degree of positively chargedamino acids in the vicinity of the MIDAS motif when compared to theother I-domains (Tulla et al 2001, Plow et al 2000). This cluster, whichappears not to be present in the other binding integrin I-domains, mayprovide α10 with specific functional characteristics thus makingalpha10beta1 unique. The I-domain of alpha10beta1 is therefore a veryinteresting target for antibody generation.

Monoclonal antibodies or fragments thereof according to the inventionmay, thus, be used for identifying, isolating, enumerating, localizing,modulating, i.e. inhibiting or stimulating, mammalian cells, e.g. ofhuman or murine origin. The cells may be e.g. mesenchymal stem cells,cells of a chondrogenic nature, chondrocytes, fibroblasts, tenocytes,myoblasts, osteoblasts, muscle cells, adipocytes, monocytes ormacrophages.

Monoclonal antibodies or fragments thereof according to the invention,such as antibody 365 or fragments thereof, may be employed in any knownanalytical or diagnostic assay or methods, e.g. different immunomethodsknown to the skilled man in the art. Examples are immunoprecipitation,immunoaffinity purification, immunoblotting, immunolocalisation,competitive binding assays, direct and indirect sandwich assays andimmunofluorescence. More examples are given in Zola 1987, and Sites etal 1982 incorporated herein by reference.

Further, the monoclonal antibody or fragments thereof may be used forvarious pharmaceutical products and for therapeutic use in mammals inthe need thereof. Such pharmaceutical products include conjugation ofmonoclonal antibodies or fragments thereof according to the invention,such as antibody 365 or fragments thereof, to different drugs known inthe art to affect, e.g. prevent, treat or alleviate, joint diseases.Examples are anti-inflammatory drugs such as non steroidalanti-inflammatory drugs (NSAIDS) for the treatment of joint diseasese.g. osteoarthritis, rheumatoid arthritis; conjugation to localanaesthetics for use post-operatively following orthopaedic surgery forthe treatment of pain management; conjugation to hypolipidemic drugs fortreatment of atherosclerotic plaque to produce a pharmaceutical productfor therapeutic use; or factors, such as growth factors, for modulatingmatrix synthesis.

As used herein, the term “fragments thereof” of a monoclonal antibodyincludes a functional portion thereof e.g., antigen binding fragmentsuch as including, but not limited to, Fv, Fab, Fab′, F(ab′)₂ fragments,single chain antibodies, and chimeric, humanized or primatized(CDR-grafted) antibodies, as well as chimeric or CDR-grafted singlechain antibodies, and the like, comprising portions derived fromdifferent species, are also encompassed by the present invention and theterm “antibody and fragments thereof”. Such fragments can be produced byenzymatic cleavage or by recombinant techniques. For instance, papain orpepsin cleavage can generate Fab or F(ab′)₂ fragments, respectively.Antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons have been introducedupstream of the natural stop site. For example, a chimeric gene encodinga F(ab′)₂ heavy chain portion can be designed to include DNA sequencesencoding the CH.sub.1 domain and hinge region of the heavy chain. Thevarious portions of these antibodies can be joined together chemicallyby conventional techniques, or can be prepared as a contiguous proteinusing genetic engineering techniques.

Murine-derived monoclonal antibodies may cause an immunogenic responsein human patients, reducing their therapeutic efficacy. To circumventthis problem, humanised antibodies have therefore been developed inwhich the murine antigen binding variable domain is coupled to a humanconstant domain. (Morrison et al (1984), Boulianne et al (1984),Neuberger et al (1985)).

To minimise the use of heterologous sequences in human antibodies thatmay cause an immunological response in a human, the CDRs or CDRsequences of murine antibodies are grafted onto the human variableregion framework (Fr) see e.g. Jones et al 1986, Riechmann et al 1988,Verhoeyen et al 1988 incorporated herein by reference. The therapeuticefficacy of this approach has been demonstrated previously by e.g.Reichmann et al (1988) and Hale et al (1989), both incorporated hereinby reference.

The term “humanized immunoglobulin” as used herein refers to animmunoglobulin comprising portions of immunoglobulins of differentorigin, wherein at least one portion is of human origin. Efficientprocedures for constructing humanized antibodies have been developed—seeFunaro et al 1996, Vaughan et al 1998, both incorporated herein byreference. Accordingly, the present invention relates to a humanizedimmunoglobulin which binds the I-domain of mammalian integrinalpha10beta1, said immunoglobulin comprising an antigen-binding regionof non-human origin, e.g., rodent such as murine, and at least a portionof an immunoglobulin of human origin e.g., a human framework region, ahuman constant region or portion thereof. For example, the humanizedantibody can comprise portions derived from an immunoglobulin ofnon-human origin with the requisite specificity, such as a mouse, andfrom immunoglobulin sequences of human origin e.g., a chimericimmunoglobulin, joined together chemically by conventional techniques,e.g., synthetic, or prepared as a contiguous polypeptide using geneticengineering techniques, e.g., DNA encoding the protein portions of thechimeric antibody can be expressed to produce a contiguous polypeptidechain.

Another example of a humanized immunoglobulin of the present inventionis an immunoglobulin containing one or more immunoglobulin chainscomprising a CDR of non-human origin e.g., one or more CDRs derived froman antibody of non-human origin, and a framework region derived from alight and/or heavy chain of human origin, e.g., CDR-grafted antibodieswith or without framework changes. In one embodiment, theantigen-binding region of the humanized immunoglobulin is derived fromthe antibody 365, disclosed in the present invention comprising CDR1,CDR2 and CDR3 of the heavy and light chain of a human antibody. Chimericor CDR-grafted single chain antibodies are also encompassed by the termhumanized immunoglobulin.

Humanized immunoglobulins can be produced using synthetic and/orrecombinant nucleic acids to prepare genes, e.g., cDNA, encoding thedesired humanized chain. For example, nucleic acid, e.g., DNA, sequencescoding for humanized variable regions can be constructed using PCRmutagenesis methods to alter DNA sequences encoding a human or humanizedchain, such as a DNA template from a previously humanized variableregion—see e.g., Kamman, M., et al., Nucl. Acids Res., 17: 5404 (1989));Sato, K., et al., Cancer Research, 53: 851-856 (1993); Daugherty, B. L.et al., Nucleic Acids Res., 19 (9): 2471-2476 (1991); and Lewis, A. P.and J. S. Crowe, Gene, 101: 297-302 (1991) all incorporated herein byreference. Using these or other suitable methods, variants can also bereadily produced. In one embodiment, cloned variable regions can bemutagenized, and sequences encoding variants with the desiredspecificity can be selected, e.g., from a phage library; see e.g.,Krebber et al., U.S. Pat. No. 5,514,548; Hoogenboom et al., WO 93/06213,published Apr. 1, 1993) all incorporated herein by reference.

Nucleic acids encoding a chimeric or humanized chain can be expressed toproduce a contiguous protein. See, e.g., Cabilly et al., U.S. Pat. No.4,816,567; Cabilly et al, European Patent No. 0,125,023 B1; Boss et al.,U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1;Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al., EuropeanPatent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter,European Patent No. 0,239,400 B1; and Queen et al., U.S. Pat. Nos.5,585,089, 5,698,761 and 5,698,762. See also, Newman, R. et al.,BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody, andLadner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science,242: 423-426 (1988)) regarding single chain antibodies, all incorporatedherein by reference.

In one embodiment of this invention the framework regions or CDRs or CDRsequences encoding fragments of antibodies according to the invention,such as antibody 365, are substituted into a suitable human antibody.

In addition, functional fragments, i.e. antigen binding fragments ofantibodies, including fragments of chimeric, humanized, primatized orsingle chain antibodies, can also be produced. Functional fragments ofthe foregoing antibodies retain at least one binding function and/ormodulation function of the full-length antibody from which they arederived. Preferred functional fragments retain an antigen-bindingfunction of a corresponding full-length antibody, i.e., the ability tobind the I-domain of mammalian integrin alpha10 or integrinalpha10beta1. Particularly preferred functional fragments retain theability to inhibit one or more functions characteristic of the I-domainof mammalian integrin alpha10 or integrin alpha10beta1, such as abinding activity, a signalling activity, and/or stimulation of acellular response. For example, in one embodiment, a functional fragmentmay modulate, i.e. inhibit or stimulate, the interaction of the I-domainof mammalian integrin alpha10 or integrin alpha10beta1 with one or moreof its ligands e.g., a cell matrix ligand such as an extracellularmatrix molecule, e.g. collagen types I-VI, IX, X, XI and/or otherextracellular matrix proteins such as chondroadherin and otherleucine-rich repeat proteins (LRR proteins), matrilin, laminin, andtenascin and/or can modulate one or more receptor-mediated functions,such as regulation of collagen turnover, regulation of matrixmetalloproteinase expression, regulation of ECM molecule turnover.

Humanisation of the mouse monoclonal antibodies or fragments thereofaccording to the invention, such as antibody 365 or fragments thereof,may, for example, be performed in the following manner:

-   -   1) RNA is harvested from mouse hybridoma clone of the present        invention.    -   2) PCR primers that hybridise to the 5′ ends of the mouse leader        sequences and to the 5′ prime ends of the mouse constant regions        are designed for cloning the kappa light chain variable regions        and heavy chain variable regions.    -   3) Complementary DNA (cDNA) is synthesised from total RNA,        followed by PCR amplification with light and heavy chain        specific primers.    -   4) Positive bacterial colonies containing mouse monoclonal        antibody variable regions are screened.    -   5) Cloned mouse monoclonal antibody leader-variable regions are        modified at the 5′- and 3′-ends, using PCR primers to create        restriction enzyme sites for insertion into expression vectors        to incorporate sequences for efficient eukaryotic translation,        and to incorporate splice-donor sites for RNA splicing of the        variable and constant regions.    -   6) The adapted mouse monoclonal antibody light and heavy chain        leader-variable regions are inserted into vectors containing,        for example, human cytomegalovirus enhancer and promoter for        transcription, a human light or heavy chain constant region, a        neomycin gene for selection of transformed cells, and the simian        virus 40 origin of replication in COS cells.

Said vectors are designed to express chimeric or reshaped human lightand heavy chains in mammalian cells. The design and construction of anengineered human antibody requires analysis of the primary amino acidsequences of the mouse monoclonal antibody variable regions furtherdescribed below to identify the residues most critical in forming theantigen-binding site.

The mouse CDRs are then joined to the FRs from selected human variableregions to form a humanised antibody.

The Primary Amino Acid Sequence of Monoclonal Antibody

The design and construction of an engineered human antibody or fragmentsthereof requires analysis of the primary amino acid sequences. Derivingthe DNA sequence, and thereby the primary amino acid sequence, of anantibody produced by a hybridoma is as of today easily done for theskilled artisan. The information retrieved from the mouse monoclonalantibody variable regions, such as the antibody 365, is used to identifythe residues most critical in forming the antigen-binding site of saidantibody.

Thus, nucleic acids encoding the heavy and/or light chains of theantibodies or portions thereof can be obtained and used in accordancewith recombinant DNA techniques for the production of the specificimmunoglobulin, immunoglobulin chain, or variants thereof, e.g.,humanized immunoglobulins, in a variety of host cells or in an in vitrotranslation system. For example, the nucleic acids, including cDNAs, orderivatives thereof encoding variants such as a humanized immunoglobulinor immunoglobulin chain, can be placed into suitable prokaryotic oreukaryotic vectors, e.g., expression vectors, and introduced into asuitable host cell by an appropriate method, e.g., transformation,transfection, electroporation, infection, such that the nucleic acid isoperably linked to one or more expression control elements, e.g., in thevector or integrated into the host cell genome.

As used herein “recombinant expression vector”, or “expression vector”refers to a transcriptional unit comprising an assembly of (1) a geneticelement or elements having a regulatory role in gene expression, forexample, promoters or enhancers, (2) a structural or coding sequencewhich is transcribed into mRNA and translated into protein, and (3)appropriate transcription initiation and termination sequences.Structural units intended for use in eukaryotic expression systemspreferably include a leader sequence enabling extracellular secretion oftranslated protein by a host cell. Alternatively, where recombinantprotein is expressed without a leader or transport sequence, it mayinclude an N-terminal methionine residue. This residue may or may not besubsequently cleaved from the expressed recombinant protein to provide afinal product

For production, host cells can be maintained under conditions suitablefor expression e.g., in the presence of inducer, suitable mediasupplemented with appropriate salts, growth factors, antibiotic,nutritional supplements, etc., whereby the encoded polypeptide isproduced. If desired, the encoded protein can be recovered and/orisolated, e.g., from the host cells, or medium, or milk. It will beappreciated that the method of production encompasses expression,transient or constantly, in a host cell of a transgenic animal.

The following is an example of a method for sequencing a mousemonoclonal antibody, such as the antibody 365, further described byJarrin and Andrieux (1999) incorporated herein by reference. Briefly:

-   -   1) RNA is extracted from the hybridoma cell line of the present        invention by standard methods, for example the method of        Gough (1988) incorporated herein by reference.    -   2) Reverse transcriptase is performed by incubating RNA with        oligonucleotide primers capable of binding specifically to each        of the immunoglobulin heavy and light chains of murine        antibodies, or by general oligo d(T)-primers.    -   3) PCR is performed to amplify the cDNA and the amplification        products analysed on an agarose gel.    -   4) PCR products corresponding to the variable region of each of        the immunoglobulin chains are digested with restriction enzymes        such as BmaH1/EcoR1 for the light chains and Pst1/Cla1 for the        heavy chains.    -   5) A vector, for example pBlueScript, is digested with        restriction enzymes corresponding to those necessary for cloning        of each chain of the mouse monoclonal antibody.    -   6) Digested immunoglobulin chains are incubated with digested        vector and ligation performed.    -   7) Electrocompetent bacteria, such as DH5alpha bacteria, are        transformed with the ligation product by electroporation.    -   8) Bacteria are selected on LB agar plates containing, for        example, ampicillin, X-Gal and IPTG. Only efficiently        transformed bacteria plus vector insert result in white        colonies.    -   9) Plasmid DNA is purified is sequencing performed using an        appropriate kit. Amino acid sequences of both the heavy and        light variable regions of the antibody can thus be deduced from        the nucleotide sequences determined above.

Modulation of Cells Using Antibody or Fragment Thereof

The monoclonal antibody or fragment thereof according to the invention,such as the antibody 365, may be used to modulate the activity of cellsexpressing alpha10beta1, as described in the paragraphs above. By“modulate activity of cells” it is further intended to mean activatingthe function or biological activity of alpha10beta1, or inhibiting bye.g. partial or complete blocking or neutralizing, thereby substantiallyinhibiting or eliminating the function or biological activity ofalpha10beta1. Typically a blocking or neutralizing antibody or fragmentsthereof will inhibit the binding of alpha10beta1 to a cell matrix ligandsuch as collagen types I-VI, IX, X, XI and/or other extracellular matrixmolecules such as chondroadherin and other leucine-rich repeat proteins(LRR proteins), matrilin, laminin, and tenascin.

Cells to be modulated are cells expressing the integrin alpha10beta1 andmay be, but are not limited to, mesenchymal stem cells, embryonic stem(ES) cells, chondrocytes, fibroblasts, adipocytes, muscle cells,tenocytes, myoblasts, osteoblasts, monocytes and macrophages.

In inhibiting the binding of extracellular matrix molecules themonoclonal antibody may induce the cell to which it binds to stimulatethe expression and/or synthesis of one or more factors such as growthfactors, cytokines, transcription factors, and/or ECM molecules.

Modulation is generally achieved by incubating the cell of interest, invivo or in vitro, with a monoclonal antibody, such as antibody 365, inempirically determined amounts. The effect is then assayed or determinedin a suitable way. In vitro a typical concentration can range from 0.1μg/ml-100 μg/ml, however, other concentration regimens may be useful andare not excluded. In vivo, depending on the type and severity of thedisease in question, about 0.015 to 15 mg of antibody or a fragmentthereof /Kg of patient weight is an initial candidate dosage foradministration to the patient.

Use of Antibody or a Fragment Thereof in ELISA Assay

Competitive binding assays rely on the ability of a labelled standard,which may be alpha10beta1 or an immunologically reactive portion thereofsuch as the I-domain, to compete with the test sample analyte, i.e.alpha10beta1 or alpha10, for binding of a limited amount of antibody.The amount of alpha10 or alpha10beta1 in the test sample e.g. humanblood, human synovial fluid, fluid surrounding the tendon, i.e.tenosynovial fluid, is then assayed as inversely proportional to theamount of standard that becomes bound to the antibodies.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion or epitope, of the protein tobe detected. In a sandwich assay, the test sample analyte is bound by afirst antibody or fragments thereof which is immobilised on a solidsupport, and thereafter a second antibody or fragments thereof bind tothe analyte, thus forming an insoluble three-part complex. The secondantibody or fragments thereof may itself be labelled with a detectablemoiety in a direct sandwich assay, or may be measured using ananti-immunoglobulin antibody or fragments thereof that is labelled witha detectable moiety in an indirect sandwich assay. For example, one typeof sandwich assay is an ELISA (enzyme-linked immunosorbent assay), inwhich case the detectable moiety is an enzyme.

A monoclonal antibody or fragment thereof according to the invention maybe used in such assays. One example of monoclonal antibody according tothe invention to be used is the antibody 365.

Use of MSCs Isolated Using an Antibody Binding to the Extracellular Partof the I-Domain of Integrin Alpha10Beta1 or a Fragment Thereof

In an additional aspect, the present invention is directed to variousmethods and uses of utilizing ES cells, MSCs, MSCs with a chondrogenicnature or chondrocytes, or other progenitor cells expressingalpha10beta1 of mammalian, such as murine or human, origin and amonoclonal antibody binding to the extracellular part of the I-domain ofintegrin alpha10beta1 or a fragment thereof, such as e.g. the antibody365 or a fragment thereof, produced by the present invention fortherapeutic and/or diagnostic purposes. For example, human MSCs orprogenitor cells may find use in:

-   -   1) regenerating mesenchymal tissues that have been damaged        through acute injury, abnormal genetic expression or acquired        disease.    -   2) treating a host with damaged mesenchymal tissue by removal of        small aliquots of e.g. bone marrow or any other tissue including        MSC, isolation of their MSCs and treatment of the damaged tissue        with MSCs combined with a biocompatible carrier suitable for        delivering the MSCs to the damaged tissue site(s).

Compositions according to the present invention, which contain MSCs, orMSCs with a chondrogenic nature, or chondrocytes, are especially usefulfor facilitating repair, reconstruction and/or regeneration of aconnective tissue defect. Connective tissue, as used herein, includescartilage, bone, ligament, tendon, stroma and muscle. Connective tissuedefects include any damage or irregularity compared to normal connectivetissue, which may occur due to trauma, disease, age, birth defect,surgical intervention etc. The use of antibodies according to theinvention disclosed herein, such as e.g. the antibody 365, areespecially suitable for use in orthopaedic surgical procedures.

Use of Chondrocytes Isolated Using a Monoclonal Antibody Binding to theExtracellular Part of the I-Domain of Integrin Alpha10Beta1 or aFragment Thereof

In an additional aspect, the present invention is directed to variousmethods of utilizing the chondrocytes and the monoclonal antibodybinding to the extracellular part of the I-domain of integrinalpha10beta1 or a fragment thereof produced for therapeutic and/ordiagnostic purposes. For example, human chondrocytes may find use in:

-   -   1) regenerating cartilage that has been damaged through acute        injury, abnormal genetic expression or acquired disease.    -   2) treating a host with damaged chondrocytes by removal of small        cartilage biopsies, isolation of the chondrocytes, culture of        the chondrocytes in vitro and reintroduction of the expanded        chondrocytes into the human patient at the site(s) of cartilage        damage.

Cartilage defects include any damage or irregularity compared to normalcartilage tissue, which may occur due to trauma, mechanical loading,disease, age, birth defect, surgical intervention etc. The use of amonoclonal antibody binding to the extracellular part of the I-domain ofintegrin alpha10beta1 or a fragment thereof, such as the antibody 365 orfragments thereof, herein is especially suitable for use in orthopaedicsurgical procedures.

Use of Embryonic Stem Cells Isolated Using Antibody Binding to theExtracellular Part of the I-Domain of Integrin Alpha10Beta1 or aFragment Thereof

In an additional aspect, the present invention is directed to variousmethods of utilizing the ES cells and the monoclonal antibody binding tothe extracellular part of the I-domain of integrin alpha10beta1 or afragment thereof or fragments thereof produced for therapeutic and/ordiagnostic purposes. For example, human ES cells may find use in e.g.

-   -   a) regenerating mesenchymal tissues that have been damaged        through acute injury, abnormal genetic expression or acquired        disease, and/or    -   b) treating a host with damaged mesenchymal tissues by isolation        of ES cells from the inner cell mass (ICM) of the blastocyst of        a 4-6 day old human embryo and culturing these cells in vitro on        mouse embryonic fibroblast feeder cells to allow the cells to        proliferate. Removal of growth factors or fibroblast growth        factor-2 (FGF-2) from the medium causes the cells to        differentiate at which point the population of cells expressing        the integrin alpha10beta1 can be identified by using the        antibody 365. Such cells can be combined with a biocompatible        carrier and surgically inserted into the damaged tissue site(s).

A Method for Isolating a Population of Mammalian Mesenchymal Stem Cells

According to the invention a method for isolating a population ofmammalian mesenchymal stem cells (MSCs), is disclosed. The methodcomprises the steps of:

-   -   a) providing a cell suspension comprising mammalian mesenchymal        stem cells,    -   b) contacting the cell suspension in a) with a monoclonal        antibody or fragments thereof according to the invention binding        to the extracellular domain of integrin alpha10beta1, under        conditions wherein said monoclonal antibody or fragments thereof        form an antibody-antigen complex with the extracellular domain        of integrin alpha10beta1,    -   c) separating cells binding to the monoclonal antibody or        fragments thereof in b), and optionally    -   d) recovering the cells binding to the monoclonal antibody or        fragments thereof in c) from said antibody or fragments thereof,

thereby producing a population of mammalian mesenchymal stem cells,optionally free from said antibody or fragments thereof.

The cell suspension provided in a) above, comprising mammalian MSCs maybe isolated from bone marrow, peripheral blood, cord blood, liver, bone,cartilage, muscle, perichondrium, periosteum, synovial tissue, fat orany tissue comprising MSCs. The cell suspension may further be isolatedfrom mammalian iliac crest, femora, tibiae, spine, rib or othermedullary spaces. Other sources of human MSCs include embryonic yolksac, placenta, and umbilical cord.

If the population of cells is collected from BM, only 0.01-0.001% of thestarting population, or “crude population”, are MSCs. Though, this mayvary between different donors.

In one further embodiment, the mammalian MSCs are human MSCs.

In one further embodiment, the mammalian MSCs are murine MSCs.

In one further embodiment the monoclonal antibody or fragment thereof isthe antibody 365 according to the invention.

In one further embodiment, the culture above is a culture for 2-4 weeks.

In one embodiment, the method for isolating a population of MSCs furthercomprises the steps of

-   a) collecting bone marrow aspirate (5-30 ml) from a human patient    into a syringe containing 6000 units of e.g. heparin to prevent    clotting,-   b) washing the marrow sample with e.g. Dulbecco's phosphate-buffered    saline (DPBS) or any similar saline solution, and recovering the    cells after centrifugation at 900 g, and repeating this procedure    once more.-   c) loading the cells onto 25 ml of Percoll of a density of 1.073    g/ml in a 50-ml conical tube and separating the cells by    centrifugation at 1100 g for 30 min at 20° C.,-   d) collecting the nucleated cells from the interface, diluting with    two volumes of DPBS, and collecting by centrifugation at 900 g.    Resuspending the cells counting the cells, and plating out the cells    at the required density, suitable 200,000-cells/cm²,-   e) culturing the cells in Dulbecco's modified Eagle's medium (DMEM)    or any other suitable medium (low glucose) containing 10% foetal    bovine serum (FBS). Replacing the medium at 24 and 72 hours and    every third or fourth day thereafter, and-   f) subculturing the hMSCs that grow as symmetric colonies at 10 to    14 days by treatment with 0.05% trypsin and 0.53 mM EDTA for 5 min,    rinsed from the substrate with serum-containing medium, collected by    centrifugation at 800 g for 5 min, and seeded into fresh flasks at    5000 to 6000 cells/cm².

The separation of MSCs is a selection and isolation step for separatingthe identified MSCs. Various techniques known to the skilled artisan maybe employed to separate the cells by initially removing cells dedicatedto other lineages than MSCs.

The antibody or fragments thereof according to the invention may beattached to a solid support to allow for a highly specific separation.The particular procedure for separation employed, e.g. centrifugation,mechanical separation, such as columns, membranes or magneticseparation, should maximize the viability of the fraction to becollected. Various techniques of different efficacy may be employedknown to a person skilled in the art. The particular technique employedwill depend upon efficiency of separation, cytotoxicity of themethodology, ease and speed of performance, and necessity forsophisticated equipment and/or technical skill.

Procedures for separation of MSCs from a cell suspension aided by theantibody or fragments thereof according to the invention may includemagnetic separation, using e.g. antibody-coated magnetic beads, affinitychromatography based on the antibody or fragments thereof according tothe invention, and “panning” with said antibody or fragments thereofattached to a solid matrix, e.g., a plate, or other convenienttechniques. Magnetic cell sorting are well known to a person skilled inthe art and is described in, for example, Haukanes and Kvam (1993)Biotechnology 11 (1):60-63, and Quirici et al (2002) Exp. Hematol30:783-791.

Techniques providing accurate separation include fluorescence activatedcell sorters by the use of the antibody or fragments thereof accordingto the invention, which can have varying degrees of sophistication,e.g., a plurality of colour channels, light scattering detectingchannels, impedance channels, etc. known to the skilled man in the art.

In one embodiment, a first enrichment step of MSCs in the provided cellpopulation is made. This first selection may be a negative selection ofthe MSCs, i.e. other lineage-committed cells are depleted, or removed,from the initial population of cells.

In still a further embodiment, the first enrichment is a positiveselection of MSCs that may be repeated until the desired purity of theMSCs is achieved.

Alternatively, MSC cells may be isolated from bone marrow. Stem cellsmay be isolated from human bone marrow by standard methods (Quirici etal (2002) Exp. Hematol 30 (7):783-791). Alternatively, commercial MSCmay be used (Poietics).

If isolated from bone marrow, bone marrow may be taken from healthyallogeneic bone marrow transplantation donors, collected in heparinizedtubes and layered onto Lymphoprep™ (density 1.077 g/ml, Nycomed, Norway)according to the manufactures' description. The low-density mononuclearcells (LD-MNC) are then isolated from the human bone marrow cells bycentrifugation. The LD-MNCs are washed twice in PBS and resuspended inMSCGM (mesenchymal stem cell growth medium) (Poetics, Cambrex BioScience Walkersville, Inc.).

Mesenchymal Stem Cells may then be purified from LD-MNCs by thefollowing standard methods: by adhesion to plastic (Pittenger et al(1999) Science 184:143), CD45⁻/alpha-glycophorin A⁻ (Reyes et al (2001)Blood. 98 (9):2615-25), CD105⁺ (Conrad et al. (2002). Exp Hematol. 30(8):887-95) and NGFR⁺ isolation (Quirici et al. (2002) Exp Hematol. 30(7):783-91).

A Method for Isolating a Population of Mammalian Chondrocytes

According to the invention a method for isolating a population ofmammalian chondrocytes is disclosed. The method comprises the steps of

-   -   a) providing a cell suspension comprising chondrocytes,    -   b) contacting the cell suspension in a) with a monoclonal        antibody or a fragment thereof according to the invention,        binding to the extracellular domain of integrin alpha10beta1,        under conditions wherein said monoclonal antibody or a fragment        thereof forms an antibody-antigen complex with the extracellular        I-domain of integrin alpha10beta1,    -   c) separating cells binding to the monoclonal antibody or a        fragment thereof in b), and optionally    -   d) recovering cells binding to the monoclonal antibody or a        fragment thereof in c) from said antibody or a fragment thereof,        -   thereby producing a population of mammalian chondrocytes,            optionally free from said antibody or a fragment thereof.

The cell suspension provided in a) above, comprising mammalianchondrocytes may be isolated from cartilage.

In one further embodiment, the monoclonal antibody or fragment thereofis the antibody 365 or a fragment thereof.

In one further embodiment, the mammalian chondrocytes are humanchondrocytes.

In one further embodiment, the mammalian chondrocytes are murinechondrocytes.

In one further embodiment, the method for isolating a population ofchondrocytes comprises the steps of

-   -   1) harvesting healthy cartilage from e.g. the femoral chondyle        and/or tibial plateau of a human specimen.    -   2) enzymatically digesting the cartilage with enzymes such as        pronase or hyaluronidase for 1 hour at 37° C. in cell medium        (Dulbecco's modified Eagles medium (DMEM) containing foetal calf        serum (FCS), penicillin/streptomycin and L-glutamine).    -   3) discarding the supernatant after pronase or hyaluronidase        digestion and further digesting the cartilage with collagenase        in DMEM for 3 hrs at 37° C.    -   4) allowing the digest to settle, removing the supernatant from        the digest and filtering through a 75 μM filter.    -   5) centrifuging the supernatant for 8 minutes at 1800 g and        washing the supernatant with PBS (Ca²⁺ and Mg²⁺ free) containing        5% FCS.    -   6) resuspending the washed cells in DMEM and incubating at        37° C. under an atmosphere of 5% CO₂.    -   7) redigesting the remaining tissue with collagenase in DMEM        until all the tissue has digested.    -   8) repeating step 5, pooling all chondrocytes obtained from        digestion, centrifuging and resuspending in DMEM for cell        counting.    -   9) culturing the chondrocytes in DMEM medium supplemented        accordingly.

The separation of chondrocytes is a selection and isolation step forseparating the identified chondrocytes. Various techniques may beemployed to separate the cells by initially removing cells other thanchondrocytes which do not express the other known chondrocyte markersaggrecan and collagen II.

The antibody or fragments thereof according to the invention may beattached to a solid support to allow for a highly specific separation,similar to that described in the method for isolation of MSCs above. Theparticular procedure for separation employed, e.g. centrifugation,mechanical separation, such as columns, membranes or magneticseparation, should maximize the viability of the cell fraction to becollected. Various techniques of different efficacy may be employedknown to a person skilled in the art. The particular technique employedwill depend upon efficiency of separation, cytotoxicity of themethodology, ease and speed of performance, and necessity forsophisticated equipment and/or technical skill.

In one embodiment, a first enrichment step of chondrocytes in theprovided cell population is made. This first selection may be a negativeselection of the chondrocytes, i.e. other cells not being chondrocytescells are depleted, or removed, from the initial population of cells.

In still a further embodiment, the first enrichment is a positiveselection of chondrocytes that may be repeated until the desired purityof the chondrocytes is achieved.

Alternatively, isolation of an integrin alpha10 positive cell populationfor differentiation to chondrocytes may be performed.

Integrin alpha10 positive cells can be isolated by the followingmethods:

Cells are labelled with 10 μg/ml mAb365 (alpha10 integrin receptor) for20 minutes at 4° C., washed and labelled with goat anti-mouse IgG microbeads (Miltenyi Biotec, Germany) for 20 minutes in 4° C. The alpha10positive cells were isolated by positive selection with an LS midiMACScolumn (Miltenyi Biotec, Germany). This procedure is performed accordingto the manufacturers' instructions.

In still another method LD-MNCs are taken and a CD45/alpha-glycophorin Adepletion kit (Miltenyi Biotec, Germany) is used. The negative(unmarked) cells are then labelled with 10 μg/ml mAb365 (alpha10integrin receptor) for 20 minutes at 4° C., washed and labelled withgoat anti-mouse IgG micro beads (Miltenyi Biotec, Germany) for 20minutes in 4° C. The alpha10 positive cells are then isolated bypositive selection with an LS midiMACS column (Miltenyi Biotec,Germany).

In still another method CD105 (Miltenyi Biotec, Germany) micro beads areused, expand the cells and labelled with 10 μg/ml mAb365 (alpha10integrin receptor) for 20 minutes at 4° C., washed and labelled withgoat anti-mouse IgG micro beads (Miltenyi Biotec, Germany) for 20minutes in 4° C. The alpha10 positive cells are then isolated bypositive selection with an LS midiMACS column (Miltenyi Biotec,Germany)).

In still another embodiment, a population of human chondrocytes areprovided by extraction from human articular cartilage. One way ofextracting a chondrocyte enriched population is described by Brittberget al (1994) in N. Engl. J. Med. (331:889-895). MAb365 may then be usedfor further enrichment of an alpha10-positive chondrocytic according tothe method for isolating a population of mammalian chondrocytes asdisclosed above.

Chondrocytes may be identified in further by measuring mRNA productionof collagen type II, or by measuring the actual collagen type Rproduction. mRNA may be measured by general protocols known to theskilled man in the art for RT-PCR (reverse transcriptase polymerasereaction). Specific primers for collagen type II are

For measurements of collagen content, either the total amount ofcollagen can be determined using the hydroxyproline assay (Woessner J. F1976 In: The Methodology of Connective Tissue Research. Ed: Hall D pp227-233) or collagen synthesis can be measured by radiolabelling with ³HProline (Scutt et al (1992) Anal. Biochem 203:290-294). Other similarmethods known to a person skilled in the art may also be used.

As an example, measurement of hydroxyproline content may be performed inthe following manner: Samples containing collagen (typically collagentype II) are hydrolysed in 6.0 M HCl for 16 hours at 110° C. to liberatehydroxyproline.

After neutralization each sample is diluted at least 15 times to preventthe salt concentration from influencing the assay. The samples are thendried under vacuum.

Example of One Method that May be Used:a. Samples (1-5 μg of hydroxyproline) are made up to 2.0 ml with assaybuffer.b. Add 1.0 ml of Chloramine-T reagent and stand for 20 minutes at roomtemperature.c. Add 1.0 ml of freshly prepared dimethylaminobenzaldehyde reagent andmix thoroughly.d. Incubate the tubes at 60° C. for 15 minutes and cool in tap water for5 minutes.e. Measure the absorbance at 550 nm within 45 minutes.Note: The hydrolysate may be passed over short columns of Dowex-50-x-8(H+ form, 200-400 mesh) to remove coloured material and impurities ifnecessary.

Reagents:

1. Stock buffer contains 50 g of citric acid (H₂O), 12 ml of glacialacetic acid, 120 g of sodium acetate, 3H₂O and 34 g of NaOH in 1.0 litreof solution. A few drops of toluene are added as preservative.2. Assay buffer: The stock buffer solution is diluted tenfold with H₂O.3. Chloramine-T reagent. 1.41 g of chloramine-T is dissolved in 20.7 mlof H₂O and mixed with 26 ml of n-propanol and 53.3 ml of stock buffer.(This reagent is stable at 4° C. for 2 weeks)4. Dimethylaminobenzaldehyde reagent. 15 g ofp-dimethylaminobenzaldehyde is suspended in 60 ml of n-propanol and 26ml of perchloric acid (60%) is added slowly (N.B. Use a fume hood withprotective goggles). This reagent must be freshly prepared.

Differentiation of Integrin Alpha10 Positive Cells

Articular cartilage has little or no capacity for self repair. Thereason for this low repair potential is unknown, but the lack of bloodsupply, low cell mobility due to the surrounding matrix and limitednumber of progenitor cells could be contributing factors. Tissueengineering approaches for cartilage have so far focused upon the use ofstem cells with a chondrogenic differentiation capacity such asmesenchymal stem cells that can be used in vivo to repair or generatenew cartilage (Jorgensen et al (2001) Ann Rheum Dis. 60 (4):305-309;Johnstone and Yoo (2001) Expert Opin Biol Ther. 2001 1 (6):915-21).Whilst it is well documented that MSCs have the inherent potential todifferentiate into osteogenic, chondrogenic, adipogenic and myocardiaccell lineages, there is currently no means of identifying the progenitorcell that will lead to these different lineages. Markers exist toindicate whether the cell is capable of expressing a cartilage phenotypei.e. collagen II and aggrecan, but these proteins are expressedextracellularly after synthesis, and cannot be used for isolation of achondrogenic cell type.

After identification of an alpha10 positive cell population by any ofthe above methods, it would be highly desirable to be able todifferentiate these cells to a chondrogenic phenotype and be able todistinguish between the other known phenotypes (Yoo et al (1998) J. BoneJ. Surgery Am 80:1745-1757).

The following methods, known to the skilled man in the art, (Tallhedenet al J. Bone. J. Surgery 85A (Suppl2):93-100) can therefore be used todetermine if the alpha10 positive cells identified using the mAb365antibody can be differentiated to a chondrocytes phenotype. Otherdifferentiation conditions may be used as a control.

Chondrogenic Differentiation

The cells are cultured as pellet mass in DMEM (GibcoBRL, Paisley, UK),insulin transferrin sodium selenite (Sigma, Sweden), 0.1 μMdexamethasone (Sigma, Sweden), 80 μM ascorbic acid-2-phosphate (Sigma,Sweden), 1 mg/ml linoleic acid-bovine serum albumin (Sigma, Sweden), 100U/ml Penicillin, 100 μg/ml Streptomycin (GibcoBRL, Paisley, UK) and 10ng/ml TGF-β3 (R&D Systems Europe Ltd., United Kingdom). To determine thechondrogenic differentiation the pellet cultures are tested for collagentype I and II, aggrecan and versican expression using Q-PCR.

Osteogenic Differentiation

To induce osteogenic differentiation the cells are cultured in DMEM-LG(GibcoBRL, Paisley, UK), 10% FCS (Sigma. St. Louis, Mo.), 50 μM ascorbicacid-2-phosphate (Sigma, Sweden), 0.10 μM dexamethasone (Sigma, Sweden),100 U/ml Penicillin and 100 μg/ml Streptomycin (GibcoBRL, Paisley, UK).At day 11, 2 mM beta-glycerophosphate (Sigma, Sweden) is added to theculture. The control cells are cultured without dexamethasone andbeta-glycerophosphate. The medium is changed every fourth day, duringthe 21 or 28 days of culture. The mineralization potential of theosteogenic differentiated cells are visualised by Von Kossa staining.

Adipogenic Differentiation

To induce adipogenic differentiation the cells are cultured in DMEM-LG(GibcoBRL, Paisley, UK), 10% FCS (Sigma, St. Louis, Mo.), 1 μMdexamethasone (Sigma, Sweden), 60 μM indomethacin (Sigma, Sweden), 0.5mM 3-isobutyl-methyl-xanthine (Sigma, Sweden), 5 μg/ml insulin (Sigma,Sweden), 100 U/ml Penicillin and 100 μg/ml Streptomycin (Gibco,Invitrogen). Every fourth day the cells are cultured during one day inDMEM-LG, 10% FCS (Sigma, St. Louis, Mo.), 100 U/ml Penicillin, 100 μg/mlStreptomycin (GibcoBRL, Paisley, UK) and 5 μg/ml insulin (Sigma,Sweden). The negative control cells are cultured in DMEM-LG (GibcoBRL,Paisley, UK) 10% FCS, 100 U/ml Penicillin and 100 μg/ml streptomycin(GibcoBRL, Paisley, UK). The cells are cultured for 14 days indifferentiation media, the differentiated cells contain lipid vacuolesthat can visualised with Oil Red O staining.

A Method for Isolating a Sub-Population of Mammalian ES Cells

According to the invention a method for isolating a sub-population ofmammalian ES cells is disclosed. The method comprises the steps of

-   -   a) providing a cell suspension comprising ES cells,    -   b) contacting the cell suspension in a) with a monoclonal        antibody or a fragment thereof according to the invention,        binding to the extracellular I-domain of integrin alpha10beta1,        under conditions wherein said monoclonal antibody or a fragment        thereof forms an antibody-antigen complex with the extracellular        I-domain of integrin alpha10beta1,    -   c) separating cells binding to the monoclonal antibody or a        fragment thereof in b), and optionally    -   d) recovering cells binding to the monoclonal antibody or a        fragment thereof in c) from said antibody or a fragment thereof,        thereby producing a sub-population of mammalian ES cells,        optionally free from said antibody or a fragment thereof.

The cell suspension provided in a) above, comprising mammalian ES cellsmay be isolated from the inner cell mass (ICM) of the blastocyst of a4-6 day old human embryo. Further ways of preparing ES cells aredescribed by Talts et al. (1999)

In one further embodiment, the monoclonal antibody or fragment thereofis the antibody 365.

In one further embodiment, the mammalian ES cells are human ES cells.

In one further embodiment, the mammalian ES cells are murine ES cells.

In one embodiment, the method for isolating a sub-population ofmammalian ES cells further comprises the steps of derivation andpropagation of ES cells.

Derivation and propagation of ES cells may be performed by the proceduredescribed below. Additional information can be found in Fong C. Y., andBongso A. (1999), Fong C. Y., et al., (1997), and in Solter, D. andKnowles, B. (1975) all incorporated herein by reference.

In brief, fertilised oocytes are cultured to the blastocyst stage (day 6after insemination), in sequential media, according to a standardco-culture free protocol (Fong and Bongso 1999). The zona pellucida isdigested by e.g. pronase (Sigma, St. Louis, Mo.) (Fong et al 1997). Theinner cell mass (ICM) is isolated by e.g. immunosurgery using anti-humanserum antibody (Sigma) followed by lysis with complement (LifeTechnologies, Gaithersburg, Md.) (Solter, D and Knowles, B1975).

The ICM may then be cultured on a mitomycin C mitotically inactivatedmouse embryonic fibroblast feeder layer (75,000 cells/cm2) ingelatine-coated tissue culture dishes. The culture medium may consist ofDMEM (Gibco, without sodium pyruvate, glucose 4500 mg/L) supplementedwith 20% foetal bovine serum (Hyclone, Logan, Utah), 0.1 mMbeta-mercaptoethanol, 1% non-essential amino acids, 2 mM glutamine, 50U/ml penicillin and 50 pg/ml streptomycin (Life Technologies). Duringthe isolation and early stages of ES cell cultivation, the medium may besupplemented with human recombinant leukaemia inhibitory factor hLlF at2000 U/ml (Amrad, Melbourne, Australia).

After 6-8 days initial plating, ICM-like clumps may be removedmechanically by a micropipette from differentiated cell outgrowths andreplated on fresh feeder layer. The resulting colonies may be furtherpropagated in clumps of about 100 stem cell-like cells on a mouse feederlayer approximately every 7 days. The clumps are either dissociatedmechanically, or with a combined approach of 5 mechanical slicingfollowed by exposure to dispase (10 mg/ml, Life Technologies).

The isolated clumps may be replated on a fresh human/mouse fibroblastfeeder layer.

In the absence of feeder cells, a colony with the typical morphology ofprimate pluripotent stem cells may be developed after 2 weeks inculture.

A Monoclonal Antibody Binding to the Extracellular Part of the I-Domainof Integrin Alpha10Beta1 or a Fragment for Positive Selection of MSCs,ES Cells or Chondrocytes

According to the invention, a monoclonal antibody or fragments thereofdisclosed is used to identify the extracellular I-domain of the integrinalpha10 chain in the molecule comprising alpha10beta1.

In one embodiment, the antibody or fragment thereof is the monoclonalantibody 365 produced by a cell line named mAb 365 deposited at theDeutsche Sammlung von Microorganismen und Zellkulturen under theaccession number DSM ACC2583. A monoclonal antibody or fragments thereofaccording to the invention has a number of advantages over a polyclonalantibody. Monoclonal antibodies are available in an unlimited supply andhigh-affinity monoclonal antibodies can bind to a large proportion ofthe available antigen. Because all the antibodies are identical and bindto the same epitope, all of the antigen interactions can be broken undersimilar conditions. Polyclonal antibodies usually bind to numerous siteson an antigen and therefore bind with high avidity. If a polyclonalantibody or fragments thereof is coupled to a column for use in aseparation procedure, the high avidity means that the antigen can bedifficult to elute. The harsh conditions required to elute the antigenmay damage the column or at least partially denature the antigen. Use ofthe monoclonal antibody or fragments thereof according to the invention,such as the antibody 365, therefore circumvents these problems.

The positive selection, e.g. a purification, may be achieved byconjugation of the monoclonal antibody according to the invention orfragments thereof to a suitable solid-phase matrix such as Protein A orProtein G, or by coupling to beads, such as magnetic beads or agarosebeads. Conjugation means for separation are known to the skilledartisan. Protocols are described in e.g. Harlow and Lane 1999,incorporated herein by reference.

Furthermore the monoclonal antibody the antibody 365 or a fragmentthereof may be coupled to magnetic beads in suspension; biotinylatedwith biotin and coupled to an avidin or streptavidin and/or coupled to asuitable support; or labelled with a fluorescent marker for use in afluorescent activated cell sorter (FACS) to allow for ease of separationof the cell type in question. Any technique may be employed which is notunduly detrimental to the viability of MSCs, ES cells or chondrocytes.

In one embodiment separation is for mammalian MSCs.

The separation, including identification and selection, may be performedby fluorescent cell sorting, by using e.g. a fluorescence activated cellsorter (FACS®) or any other methodology having high specificity.Multi-colour analyses may be employed with the FACS, which isparticularly convenient. MSCs may be separated on the basis of the levelof staining for the particular antigens. In a first separation,antibodies for other markers may be used labelled with one fluorochrome,while the antibody or a fragment thereof according to the invention maybe conjugated to different fluorochrome(s). Other markers to be used mayin further embodiments be SH-2, SH-3, CD29, CD44, CD71, CD90, CD106,CD120a, CD124, CD105, and Stro-1 that MSCs may express. Markers that arenot expressed on MSCs are CD14, CD34 and CD45 and their expression, orlack of, may in further embodiments also be evaluated together with theantibody according to the invention or a fragment thereof binding to theI-domain of integrin alpha10beta1.

If further lineages or cell populations not being MSCs are to be removedin one step, various antibodies to such lineage-specific markers may beincluded. Fluorochromes, which may find use in a multi-colour analysis,include phycobiliproteins, e.g., phycoerythrin and allophycocyanins,fluorescein, Texas red, etc. well known to the skilled man in the art.

The MSCs may be selected against dead cells, by employing dyesassociated with dead cells (propidium iodide, LDS). The cells may becollected in a medium comprising foetal calf serum.

MSCs may as well be selected based on light-scatter properties and theirexpression of various cell surface antigens, in combination with theidentification using the antibody according to the invention or afragment thereof.

In one embodiment separation is for mammalian chondrocytes.

The separation, including identification and selection, is performed byfluorescent cell sorting, by using e.g. a fluorescence activated cellsorter (FACS®) or any other methodology having high specificity.Multi-colour analyses may be employed with the FACS. Chondrocytes may beseparated on the basis of the level of staining for alpha10beta1expression. In a first separation, antibodies for other markersexpressed on non-chondrogenic cells may be used as a negative selectionstep for chondrocytes. The antibody or a fragment thereof according tothe invention may be conjugated to different fluorochrome(s) to be usedin a positive selection step.

If further lineages or cell populations not being chondrocytes are to beremoved in one step, various antibodies to such lineage specific markersmay be included. Fluorochromes, which may find use in a multi-colouranalysis, include phycobiliproteins, e.g., phycoerythrin andallophycocyanins, fluorescein, Texas red, etc. well known to a personskilled in the art.

The chondrocytes may be selected against dead cells, by employing dyesassociated with dead cells (propidium iodide, LDS) or othernon-chondrocytic cells, such as dedifferentiated cells. The chondrocytesmay be collected in a medium comprising foetal calf serum.

In one embodiment separation is for a sub-population of mammalian EScells expressing alpha10beta1.

The separation of such ES cells, including identification and selection,may be performed by fluorescent cell sorting, by using e.g. afluorescence activated cell sorter (FACS®) or any other methodologyhaving high specificity. Multi-colour analyses may be employed with theFACS. human ES cell markers include Stage-specific Embryonic Antigen-3(SSEA-3), SSEA-4, GCTM-2, alkaline phosphatase, TRA-1-60, TRA-1-81(reference Pera et al (2000); www.nih.gov/news/stemcell/scireport.htm)

Other techniques for positive or negative selection of MSCs, ES cellsand chondrocytes may be employed. The techniques used should permitaccurate separation, such as affinity columns, magnetic beads, or othertypes of beads readily conjugated with an antibody binding to theI-domain such as the antibody according to the invention, or similartypes of techniques.

While it is believed that the particular order of separation is notcritical to this invention, the order indicated in the embodiment belowis one particular embodiment.

One embodiment for positive selection of MSCs, ES cells or chondrocytesincludes that the cells in a provided cell suspension are initiallyseparated by a crude separation, such as a centrifugation, a negativeselection, or both, followed by a fine separation. The fine separationis a positive selection, using a monoclonal antibody or fragment thereofaccording to the invention, such as the antibody 365 or a fragmentthereof, binding to the I-domain of integrin alpha10beta1 on MSCs, EScells or chondrocytes. Further, a negative selection for markersassociated with cells committed to other lineages, and other stem cellpopulations not being MSCs, ES cells or chondrocytes may be included.

The isolated cell population(s) is/are further described below.

In one embodiment, the monoclonal antibody or a fragment thereofaccording to the invention, such as the antibody 365 or a fragmentthereof, used in the positive selection is linked to a solid phase.Examples of solid phases to be used are Protein A or Protein G,activated beads such as agarose beads, cross-linked agarose beads,polyacrylamide beads, copolymers of polyacrylamide and agarose beads orpolyacrylic beads.

In one embodiment the solid phase is a bead. Examples of beads are beadscomprising Protein A or Protein G, activated beads such as agarosebeads, cross-linked agarose beads, polyacrylamide beads, copolymers ofpolyacrylamide and agarose beads or polyacrylic beads. Beads areactivated with, for example Carbonyldiimadazole, Cyanogen bromide and byother similar methods well known to a skilled man in the art and furtherexemplified by Harlow and Lane, 1988, included herein by reference.

In one embodiment the solid phase is a bead such as magnetic bead. Cellscan then be sorted using magnetic cell sorting, such as the MACS®system.

In still a further embodiment, the selected and isolated mammalianmesenchymal stem cells, ES cells or chondrocytes are human cells.

In still a further embodiment, the selected and isolated mammalianmesenchymal stem cells, ES cells or chondrocytes are murine cells.

Optionally, cells binding to the monoclonal antibody or a fragmentthereof according to the invention, e.g. the antibody 365 or a fragmentthereof, may be recovered. By “recovering” it is herein intended to meanthat the selected cells are released from the monoclonal antibody or afragment thereof to which they are bound, thereby producing a populationof cells, e.g. MSCs, ES cells or chondrocytes, free from said antibodyor a fragment thereof.

Thus, a monoclonal antibody binding to the extracellular part of theI-domain of integrin alpha10beta1 or a fragment thereof, such as e.g.the antibody 365 or a fragment thereof, will be highly valuable forfurther evaluation and enrichment of the chondrocytes, ES cells or MSCpopulation.

Other Markers on MSCs

In further embodiments of the invention, the identification of MSCs maybe combined with other markers known to be expressed by MSCs. Such othermarkers are SH-2, SH-3, CD29, CD44, CD71, CD90, CD106, CD120a, CD124,CD105, and Stro-1 that MSCs may express. Markers that are not expressedon MSCs are CD14, CD34 and CD45 and their expression may in furtherembodiments also be evaluated together with the binding of the antibodyaccording to the invention or a fragment thereof.

Other Markers of Cartilage

As of today, no other cell surface markers for chondrocytes exist, asidefrom the integrin alpha10beta1. Antibodies according to the invention,reactive to the I-domain of alpha10, e.g. the antibody 365 disclosed inthe present invention, are thus unique. Markers on cartilage matrix maystill be used in combination with an antibody according to theinvention. Examples of markers of cartilage matrix are aggrecan,collagen type II and the markers disclosed in US2003/0039966 by Heringand Johnstone incorporated herein by reference.

Other Markers on ES Cells

In further embodiments of the invention, the identification of human EScells may be combined with other markers known to be expressed by humanES cells. Such other markers on human ES cells include Stage-specificEmbryonic Antigen-3 (SSEA-3), SSEA-4, GCTM-2, alkaline phosphatase,TRA-1-60, TRA-1-81 (reference Pera et al (2000);www.nih.gov/news/stemcell/scireport.htm).

A Population of Mammalian Mesenchymal Stem Cells

A population of mammalian mesenchymal stem cells are obtainable by themethod according to the invention. The population is characterised bymesenchymal stem cells that binds to a monoclonal antibody binding tothe extracellular part of the I-domain of integrin alpha10beta1 or afragment thereof, such as e.g. the antibody 365 or a fragment thereof.

In one embodiment, the mammalian stem cells are human mesenchymal stemcells.

In one further embodiment, the mammalian stem cells are murinemesenchymal stem cells.

In order to obtain human mesenchymal stem cells, it is necessary toisolate rare pluripotential mesenchymal stem cells, e.g. only one MSCsper 100 000 nucleated cells—see Bruder et al 1997 incorporated herein byreference—from other cells in the bone marrow or other MSCs sources,such as ES cells. Mammalian MSCs may be isolated from bone marrow,peripheral blood, cord blood, liver, bone, cartilage, muscle,perichondrium, periosteum, fat or any tissue comprising MSCs. The cellsuspension may further be isolated from mammalian iliac crest, femora,tibiae, spine, rib or other medullary spaces. Other sources of humanMSCs include embryonic yolk sac, placenta, umbilical cord, foetal andadolescent skin.

Said mesenchymal stem cells are the formative pluripotential blast cellsthat are capable of differentiating into any of the specific types ofmesenchymal or connective tissues, i.e. the tissues of the body thatsupport specialised elements; particularly bone, cartilage, muscle,tendon, ligament, marrow stroma, fat.

Use of an Isolated MSCs Population

A population of MSCs specifically isolated using a monoclonal antibodybinding to the extracellular part of the I-domain of integrinalpha10beta1 or a fragment thereof, such as e.g. the antibody 365 or afragment thereof, can be used for tissue repair and regeneration ofcartilage, but also for the repair of bone, muscle, tendon, andligament, either alone or immobilized to a biomaterial scaffold whichacts as a support a guidance template.

Types of scaffold include, bioresorbable poly(α-hydroxy esters)scaffolds such as polylactic acid (PLLA), polyglycolic acid (PGA) andcopolymer (PLGA).

Further embodiments include scaffolds derived from polymeric gels suchas hyaluronic acid, collagen, alginate and chitosan.

Further embodiments include scaffolds derived from porous carriers, suchas tricalcium phosphate and/or hydroxyapatite ceramic block (Luyten etal 2001)

Various procedures for transferring and immobilising the MSCs includinginjecting the isolated cells into the site of skeletal defect,incubating isolated cells optionally with the antibody 365 to hold thecells in place in suitable gel and implanting, incubating withbioresorbable scaffold etc. Thus, one embodiment is the conjugation ofthe antibody 365 to a bioresorbable scaffold allowing immobilisation ofthe cells before implantation into the damaged or defect site, e.g. intothe site of a skeletal defect. The scaffold allows 3D immobilization ofMSCs. Suitable biomaterial scaffolds are exemplified above. The examplesgiven are not limiting the use of other suitable scaffolds obvious to askilled artisan to choose if more suitable for the particularapplication.

MSCs isolated with monoclonal antibodies according to the invention orfragments thereof, such as the antibody 365, may also be directlyinjected back into the damaged site of the skeletal defect.

In still a further embodiment, injected cells are after injectioncaptured and immobilized in a biomaterial scaffold conjugated to amonoclonal antibody according to the invention and further placed intothe damaged area. The cells are thus captured and held in place at acorrect position in a damaged site.

A Population of Mammalian Chondrocytes

The expression of alpha10beta1 on the cell surface of chondrocytesprovides a useful tool for the identification and isolation ofchondrocytes. Thus, the monoclonal antibody according to the inventionor a fragment thereof is of great value in identifying chondrocytes orother cells expressing the integrin alpha10beta1 on their surface fortreatment purposes in particular for the isolation of chondrocytes andchondrogenic cells e.g. synovial cells from the synovial lining of apatient (Nishimura et al 1999) for tissue engineering.

In one embodiment, the monoclonal antibody or fragment thereof is theantibody 365 or a fragment thereof.

Using a monoclonal antibody binding to the extracellular part of theI-domain of integrin alpha10beta1 or a fragment thereof, such as e.g.the antibody 365 or a fragment thereof for isolation of chondrocytes onemay use autologous cells in procedures whereby diseased or damagedcartilage is to be repaired.

Mature articular cartilage has a poor reparative response to injury andits irreparable breakdown is a common feature of degenerative jointdiseases, such as e.g. arthritis including osteoarthritis and rheumatoidarthritis. Repair of such injuries has focused upon different tissueengineering strategies, including the use of cell transplantation usingautologous chondrocyte. Critical to these techniques is theidentification and/or isolation of chondrocytes producing a hyalinecartilage.

Thus, antibodies according to the invention may be used for isolation ofchondrocytes as well as identification of a cell with a chondrocytephenotype i.e. chondrocytes producing a hyaline cartilage, and thus theantibody or fragment thereof can be used as a quality control, before invivo implantation, to guarantee that only hyaline cartilage-producingcells are replaced into the diseased or damaged area.

Uses of Isolated Chondrocytes

Specifically, a monoclonal antibody binding to the extracellular part ofthe I-domain of integrin alpha10beta1 or a fragment thereof, such ase.g. the antibody 365 or a fragment thereof may be used to identify andisolate cells with a chondrogenic cell phenotype, particularlychondrocytes. Such a population of cells specifically isolated using theantibody 365 or fragments thereof may be used for autologous tissuerepair and regeneration of cartilage either alone or in combination withany tissue scaffolds, such as a biomaterial scaffold, as a support.

Scaffolds to be used are mentioned in the paragraphs above.

A method for autologous tissue repair using chondrocytes isolated with amonoclonal antibody binding to the extracellular part of the I-domain ofintegrin alpha10beta1 or a fragment thereof, such as e.g. 365 or afragment thereof, for autologous chondrocyte transplantation isdisclosed. Further methods are described by Brittberg et al.incorporated herein by reference. Further embodiments of the methodcomprises the steps of

-   -   a) harvesting a biopsy comprising cartilage of healthy cartilage        from a human subject, e.g. a patient, whilst undergoing an        arthroscopic procedure,    -   b) enzymatically digesting the cartilage firstly with enzymes,        such as pronase or hyaluronidase, and subsequently with        collagenases to extract a cell population comprising        chondrocytes,    -   c) culturing the cell population comprising chondrocytes in a        suitable medium, for example, DMEM, F12 etc for 2-4 weeks,    -   d) purifying the chondrocytes from the cell population after        culturing for about 2-4 weeks using an antibody according to the        invention or a fragment thereof, either by FACS, mechanical        purification such as beads, e.g. magnetic beads, or by use of a        kit further described below,    -   e) performing surgery of a human patient to expose the damaged        cartilage and at the same time remove periosteum from the medial        tibia of the same human patient. Suture the periosteal flap over        the injured or damaged cartilage area,    -   f) implanting the chondrocytes purified with the antibody or a        fragment thereof, into the joint of the same human patient.        Purified chondrocytes are injected under the periosteal flap, or        in an alternative approach,    -   g) implanting the chondrocytes in combination with a biomaterial        support (examples given previously) in which the monoclonal        antibody or fragment thereof, is coupled/conjugated in order to        immobilize the cells.

A Population of Embryonic Stem Cells

A population of mammalian embryonic stem (ES) cells are obtainable bythe method according to the invention. The population is characterisedby differentiated ES cells that bind to a monoclonal antibody binding tothe extracellular part of the I-domain of integrin alpha10beta1 or afragment thereof, such as e.g. the antibody 365 or a fragment thereof.

In one embodiment, the ES cells are human ES cells.

In one further embodiment, the mammalian stem cells are murine ES cells.Human ES cells may be derived from the inner cell mass (ICM) of theblastocyst of a 4-6 day old human embryo and may further be cultured invitro on e.g. mouse embryonic fibroblast feeder cells to allow the cellsto proliferate. Removal of growth factors or fibroblast growth factor-2(FGF-2) from the medium causes the cells to differentiate at which pointthe population of cells expressing the integrin alpha10beta1 can beidentified by using the antibody 365.

Uses of ES Cells

Specifically, a monoclonal antibody binding to the extracellular part ofthe I-domain of integrin alpha10beta1 or a fragment thereof, such ase.g. the antibody 365 or a fragment thereof may be used to identify andisolate differentiated ES cells. Such a population of cells specificallyisolated using the antibody 365 or fragments thereof may be used forautologous tissue repair and regeneration of mesenchymally-derivedtissues e.g. cartilage either alone or in combination with any tissuescaffolds, such as a biomaterial scaffold, as a support.

Scaffolds to be used are mentioned in the paragraphs above. ES cellsisolated with monoclonal antibodies according to the invention orfragments thereof, such as the antibody 365, may also be directlyinjected back into the damaged site of the skeletal defect.

In still a further embodiment, injected cells are, after injection,captured and immobilized in a biomaterial scaffold conjugated to amonoclonal antibody according to the invention and further placed intothe damaged area. The cells are thus captured and held in place at acorrect position in a damaged site.

A Method for Identifying a Mammalian MSC

A method for identifying a MSC in a sample is disclosed. The methodcomprises the steps of

-   -   a) providing a sample cell suspension comprising of a        mesenchymal stem cell,    -   b) contacting said sample cell suspension with a monoclonal        antibody or a fragment thereof according to the invention        binding to the extracellular domain of integrin alpha10beta1        produced by a cell line according to the invention,    -   c) incubating the sample cell suspension and the monoclonal        antibody or a fragment thereof under conditions wherein said        monoclonal antibody or a fragment thereof forms an        antibody-antigen complex with the extracellular domain of        integrin alpha10beta1 on a mesenchymal stem cell,    -   d) optionally adding a second labelled antibody or a fragment        thereof to the sample, wherein the second antibody or a fragment        thereof binds to the monoclonal antibody according to the        invention or a fragment thereof in b)    -   e) detecting the monoclonal antibody or a fragment thereof bound        to the extracellular domain of integrin alpha10beta1 of the        sample b), or optionally detecting the second labelled antibody        or a fragment thereof in c) bound to the monoclonal antibody or        a fragment thereof.

Mammalian MSCs may be isolated from bone marrow, peripheral blood, cordblood, liver, bone, cartilage, muscle, perichondrium, periosteum, fat orany tissue comprising MSCs. The cell suspension may further be isolatedfrom mammalian iliac crest, femora, tibiae, spine, rib or othermedullary spaces. Other sources of human MSCs include embryonic yolksac, placenta, umbilical cord, foetal and adolescent skin.

The sample cell suspension is provided from different mammals, such as ahuman being, a rodent, including all members of the phylogeneticRodentia, such as a mouse, or a rat.

The contacting of said sample cell suspension with a monoclonal antibodyaccording to the invention or a fragment thereof may be in any suitablecell culturing media, such as Dulbecco's Modified Eagle's Medium (DMEM),Hams F12 Nutrient Mixture, or in any physiological saline solution,preferably buffered, such as phosphate buffer saline (PBS), optionallywith foetal calf serum (FCS) or bovine serum albumin (BSA) present.

The incubation of the cell suspension and the monoclonal antibody or afragment thereof should be under conditions wherein said monoclonalantibody or a fragment thereof forms an antibody-antigen complex withthe extracellular domain of integrin alpha10beta1 on a mesenchymal stemcell.

A second labelled antibody or a fragment thereof optionally added to thesample may be antibodies binding to molecules known to be expressed byMSCs. Such other molecules, or markers, are SH-2, SH-3, CD29, CD44,CD71, CD90, CD106, CD120a, CD124, CD105, and Stro-1 that MSCs mayexpress. Markers that are not expressed on MSCs are CD14, CD34 and CD45and their expression may in further embodiments also be evaluated incombination with the binding of the antibody according to the inventionor a fragment thereof.

A Method for Identifying a Mammalian Chondrocyte

A method for identifying a chondrocyte in a sample is further disclosed.The method comprises the steps of

-   -   a) providing a sample cell suspension comprising of a        chondrocyte,    -   b) contacting said sample cell suspension with monoclonal        antibody according to the invention or a fragment thereof        binding to the extracellular domain of integrin alpha10beta1        produced by a cell line according to the invention,    -   c) incubating the sample cell suspension and the monoclonal        antibody or a fragment thereof under conditions wherein said        monoclonal antibody or a fragment thereof forms an        antibody-antigen complex with the extracellular domain of        integrin alpha10beta1 on a chondrocyte,    -   d) optionally adding a second labelled antibody or a fragment        thereof to the sample, wherein the second antibody or a fragment        thereof binds to the monoclonal antibody according to the        invention or a fragment thereof in b)    -   e) detecting the monoclonal antibody according to the invention        or a fragment thereof bound to the extracellular domain of        integrin alpha10beta1 of the sample b), or optionally detecting        the second labelled antibody or a fragment thereof in c) bound        to the monoclonal antibody or a fragment thereof.        The Sample Cell Suspension May be Isolated from Cartilage.

The sample cell suspension is provided from different mammals, such as ahuman being, a rodent, including all members of the phylogeneticRodentia, such as a mouse, or a rat.

The contacting of said sample cell suspension with a monoclonal antibodyaccording to the invention or a fragment thereof may be in any suitablecell culturing media, such as Dulbecco's Modified Eagle's Medium (DMEM),Hams F12 Nutrient Mixture, or in any physiological saline solution,preferably buffered, such as phosphate buffer saline (PBS), optionallywith foetal calf serum (FCS) or bovine serum albumin (BSA) present.

The incubation of the cell suspension and the monoclonal antibody or afragment thereof should be under conditions wherein said monoclonalantibody or a fragment thereof forms an antibody-antigen complex withthe extracellular domain of integrin alpha10beta1 on a chondrocyte

A second labelled antibody or a fragment thereof optionally added to thesample may be antibodies binding to molecules known to be expressed bycartilage matrix antibody according to the invention as previouslymentioned.

A Method for Identifying a Sub-Population of Mammalian ES Cells

A method for identifying a sub-population of mammalian ES cells in asample is disclosed. The method comprises the steps of

-   -   a) providing a sample cell suspension comprising of a        differentiated ES cell,    -   b) contacting said sample cell suspension with a monoclonal        antibody or a fragment thereof according to the invention        binding to the extracellular domain of integrin alpha10beta1        produced by a cell line according to the invention,    -   c) incubating the sample cell suspension and the monoclonal        antibody or a fragment thereof under conditions wherein said        monoclonal antibody or a fragment thereof forms an        antibody-antigen complex with the extracellular domain of        integrin alpha10beta1 on a differentiated ES cell,    -   d) optionally adding a second labelled antibody or a fragment        thereof to the sample, wherein the second antibody or a fragment        thereof binds to the monoclonal antibody according to the        invention or a fragment thereof in b)    -   e) detecting the monoclonal antibody or a fragment thereof bound        to the extracellular domain of integrin alpha10beta1 of the        sample b), or optionally detecting the second labelled antibody        or a fragment thereof in c) bound to the monoclonal antibody or        a fragment thereof.

The sample cell suspension may be isolated from the inner cell mass(ICM) of the blastocyst of a 4-6 day old human embryo.

The sample cell suspension is provided from different mammals, such as ahuman being, a rodent, including all members of the phylogeneticRodentia, such as a mouse, or a rat.

The contacting of said sample cell suspension with a monoclonal antibodyaccording to the invention or a fragment thereof may be in any suitablecell culturing media, such as Iscove's modified Dulbecco's medium(IMDM), or in any physiological saline solution, preferably buffered,such as phosphate buffer saline (PBS), optionally with foetal calf serum(FCS) or bovine serum albumin (BSA) present.

The incubation of the cell suspension and the monoclonal antibody or afragment thereof should be under conditions wherein said monoclonalantibody or a fragment thereof forms an antibody-antigen complex withthe extracellular domain of integrin alpha10beta1 on a differentiated EScell.

A second labelled antibody or a fragment thereof optionally added to thesample may be antibodies binding to molecules known to be expressed byES cells. Such other molecules, or markers, include Stage-specificEmbryonic Antigen-3 (SSEA-3), SSEA-4, GCTM-2, alkaline phosphatase,TRA-1-60, TRA-1-81.

Markers that are not expressed on ES cells are CD14, CD34 and CD45 andtheir expression may in further embodiments also be evaluated incombination with the binding of the antibody according to the inventionor a fragment thereof.

A Method for Detecting the Expression of Integrin Alpha10Beta1 in aTissue Sample

A method for detecting the expression of integrin alpha10beta1 in atissue sample is disclosed. The method comprises the steps of

-   -   a) providing a tissue sample,    -   b) providing monoclonal antibody according to the invention or a        fragment thereof binding to the extracellular domain of integrin        alpha10beta1 produced by a cell line according to claim 1,    -   c) incubating the tissue sample and the monoclonal antibody or a        fragment thereof under conditions wherein said monoclonal        antibody or a fragment thereof forms an antibody-antigen complex        with the extracellular domain of integrin alpha10beta1,    -   d) optionally adding a second labelled antibody or a fragment        thereof to the sample, wherein the second antibody or a fragment        thereof binds to the monoclonal antibody according to the        invention or a fragment thereof in b),    -   e) detecting the monoclonal antibody according to the invention        or a fragment thereof bound to the extracellular domain of        integrin alpha10beta1 of the sample b), or optionally detecting        the second labelled antibody or a fragment thereof in c) bound        to the monoclonal antibody or a fragment thereof.

A Method for In Vivo Imaging the Expression of Integrin Alpha10Beta1 ina Mammal

A method for in vivo imaging the expression of integrin alpha10beta1 ina mammal is disclosed. By imaging the expression, distribution andquantification of alpha10beta1 can be determined The method comprisesthe steps of

-   -   a) providing a mammal,    -   b) providing a monoclonal antibody or a fragment thereof binding        to the extracellular domain of integrin alpha10beta1 produced by        a cell line according to claim 1,    -   c) administering the monoclonal antibody or a fragment thereof        to the mammal so as to allow the antibody or a fragment thereof        to bind to the extracellular domain of integrin alpha10beta1 of        cells in said mammal,    -   d) optionally adding a second labelled antibody or a fragment        thereof to the sample, wherein the second antibody or a fragment        thereof binds to the monoclonal antibody or a fragment thereof        in c),    -   e) detecting the monoclonal antibody or a fragment thereof bound        to the extracellular domain of integrin alpha10beta1 of said        cells in c), or optionally detecting the second labelled        antibody or a fragment thereof in d) bound to the monoclonal        antibody or a fragment thereof, and    -   f) creating an image of the detected antibody or a fragment        thereof,

thereby imaging the expression of integrin alpha10beta1 on cells in amammal in vivo.

In one embodiment, said antibody or a fragment thereof is labelled witha detectable moiety, such as a radio-opaque agent or radioisotope.

The monoclonal antibody or a fragment thereof in c) above must beadministered so as to allow the antibody or a fragment thereof to bindto the extracellular domain of integrin alpha10beta1 of cells in saidmammal. Administration may be performed by injection into thebloodstream, e.g. intravenous, into synovial fluid, intramuscular,intraperitoneal, intra-articular, subcutaneous, into the cavitysurrounding the tendon or directly into a plaque formed in a bloodvessel. The presence and location of a labelled antibody or a fragmentthereof in a host is assayed by e.g. imaging.

Information obtained by imaging, using the method described above, isuseful when studying the progression, regression or repair during amedical treatment of e.g. a joint disease, such as e.g. arthritisincluding osteoarthritis and rheumatoid arthritis. Other diseases aretendinitis, e.g. peritendinitis, tenosynovitis, insertitis, tendinousbursitis and apophysitis, and in atherosclerosis e.g. the detection ofatherosclerotic plaque in blood vessels.

The antibody according to the invention, such as the antibody 365, or afragment thereof may be labelled with any moiety or means that isdetectable in a host. Suitable means for detection are any non-invasivemethods in vivo, such as any imaging method. Examples of such methodsare Magnetic Resonance Imaging (MRI), Ultrasound, such as intravascularultrasound (IVUS), Computed tomography, such as Electron Beam ComputedTomography (EBCT) and multislice tomographic scanning, as well asangiography. Any other suitable means known to the skilled man in theart may also be used such as means described in Narayanan et al 2000,included herein by reference.

A Composition

According to the invention, a composition comprising a monoclonalantibody binding to the extracellular part of the I-domain of integrinalpha10beta1 or a fragment thereof, such as e.g. the antibody 365 or afragment thereof produced by a hybridoma cell line according to theinvention is disclosed.

In a further embodiment, the monoclonal antibody according to theinvention or a fragment thereof is conjugated. Any known method in theart for separately conjugating the antibody or a fragment thereof to thedetectable moiety may be employed including those methods described byDavid et al (1974), Pain et al (1981) and Nygren et al (1982).

For diagnostic applications, the monoclonal antibody according to theinvention or a fragment thereof will typically be conjugated andlabelled with a detectable moiety. The detectable moiety can be any onethat is capable of producing, either directly or indirectly, adetectable signal.

In one embodiment, said monoclonal antibody or a fragment thereof isfurther conjugated and comprises a detectable label, such as afluorescent or chemiluminescent compound, such as fluorochromes, e.g.fluoroscein isothiocyanate, rhodamine, or luciferine, or anyfluorochrome which may find use in a multi-colour analysis includingphycobiliproteins, e.g., phycoerythrin and allophycocyanins,fluorescein, Texas red, etc. well known to a person skilled in the art.Fluorochromes can be used with a fluorescence activated cell sorter; orthe like, to allow for ease of separation of the particular cell type.

In a further embodiment the monoclonal antibody or a fragment thereofcan be conjugated, or labelled, with a suitable radioactive or enzymaticlabel by conventional methods and/or bound to suitable solid phasesknown to the skilled man in the art. Examples of enzymes are alkalinephosphatase, beta-galactosidase or horseradish peroxidase, aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²⁵I or radioactive isotopiclabels that are useful within the body of a human subject and include¹¹¹I, ⁹⁹Tc, ⁶⁷Ga, ¹⁸⁶Re, and ¹³²I.

In a further embodiment, said monoclonal antibody or a fragment thereoffurther comprises means for separation of a cell, which allows fordirect or indirect separation e.g. biotin, binding to avidin; orstreptavidin. The means for separation may be bound to a solid supportsuch as beads, e.g. magnetic beads, agarose or other similar types ofbeads known to the skilled man in the art. Any means suitable forseparation of cells may be employed on the condition that the separationis not unduly detrimental to the viability of a cell.

In a further embodiment the monoclonal antibody or a fragment thereofcan be used in combination with, or coupled to, an immunochemical suchas biotin and its analogues (e.g. iminobiotin), avidin and its analogues(streptavidin), alkaline phosphatases or other such markers for theidentification and/or quantification of MSCs, ES cells or chondrocytesand the direct/indirect separation of said cells.

Medical Use

A use of a monoclonal antibody or a fragment thereof binding to theextracellular domain of integrin alpha10beta1 produced by a cell lineaccording to the invention, such as the antibody 365, for thepreparation of a pharmaceutical composition for the treatment of jointdiseases, such as e.g. arthritis including osteoarthritis and rheumatoidarthritis in a mammal in the need thereof is disclosed. Other diseasesare tendinitis, e.g. peritendinitis, tenosynovitis, insertitis,tendinous bursitis and apophysitis, and atherosclerosis.

In a further embodiment, an additional pharmaceutically acceptable drugaffecting joint diseases, such as e.g. arthritis includingosteoarthritis and rheumatoid arthritis is included to thepharmaceutical composition. Other diseases are tendinitis, e.g.peritendinitis, tenosynovitis, insertitis, tendinous bursitis andapophysitis, and atherosclerosis.

A monoclonal antibody binding to the extracellular part of the I-domainof integrin alpha10beta1 or a fragment thereof, such as e.g. theantibody 365 or a fragment thereof is characterized as having theability to specifically immunoreact with the I-domain of the alphasubunit of the integrin alpha10beta1 and thereby inhibit the capacity ofthe integrin to specifically bind to its ligand by an interaction with aligand-containing protein. Thus the antibody or fragment thereof isuseful to inhibit and stimulate, and thereby modulate, either in vivo orin vitro, the functionality of the cells that contain integrinalpha10beta1 with which the antibody or a fragment thereof immunoreacts.

For treatment and therapeutic applications, the antibody or a fragmentthereof is administered to a mammal, preferably human, in apharmaceutically acceptable dosage form. The antibody or a fragmentthereof may be administered intravenously as a bolus, or by continuousinfusion over a period of time, by intramuscular, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical or inhalationroutes.

An administration vehicle comprising a monoclonal antibody or a fragmentthereof in a dosage form binding to the extracellular domain of integrinalpha10beta1 produced by a cell line according to the invention,pharmaceutical acceptable carrier, and a pharmaceutical acceptable drugaffecting joint diseases, such as e.g. arthritis includingosteoarthritis and rheumatoid arthritis is disclosed. Other diseases aretendinitis, e.g. peritendinitis, tenosynovitis, insertitis, tendinousbursitis and apophysitis, and atherosclerosis.

The dosage forms encompass pharmaceutically acceptable carriers that areinherently non-toxic and non-therapeutic. Examples of such carriersinclude ion exchangers, alumina, aluminium stearate, lecithin, serumproteins such as human serum albumin, buffers such as phosphate orglycine, sorbic acid, potassium sorbate, partial glyceride mixtures ofsaturated vegetable fatty acids, water, salts, or electrolytes such asprotamine sulphate, sodium chloride, metal salts, colloidal silica,magnesium trisilicate, polyvinyl pyrrolidone, cellulosic polymers andpolyethylene Glycol. Carriers for topical or gel-based forms of antibodyor a fragment thereof include polysaccharides such as sodiumcarboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers,polyethylene glycol and wood wax alcohols. Conventional depot formsinclude, for example, liposomes, microcapsules, nano-capsules, plasters,sublingual tablets, and polymer matrices such as poly(orthoesters),polylactide:polyglycolide polymers.

When the antibody or a fragment thereof is present in an aqueous dosageform, rather than being lyophilised, the antibody or a fragment thereoftypically may be formulated at a concentration of about 0.1 mg/ml to 100mg/ml, although a wide variation outside of these ranges is permitted.

For the prevention or treatment of disease, the appropriate dosage ofthe antibody 365 will depend on the type of disease to be treated, theseverity and course of the disease, whether the antibody or a fragmentthereof is administered for preventative or therapeutic purposes, thecourse of previous therapy and the patient's clinical history andresponse to the antibody or a fragment thereof. The antibody or afragment thereof is suitably administered to the patient at one time orover a series of treatments.

Depending on the type and severity of the disease, about 0.015 to 15 mgof antibody or a fragment thereof /Kg of patient weight is an initialcandidate dosage for administration to the patient. Administration maybe, for example, by one or more separate administrations, or bycontinuous infusion. For repeated administrations over several days orlonger, depending on the condition, the treatment is repeated until adesired suppression or alleviation of the disease symptoms occurs.However, other dosage regimens may be useful and are not excluded.

According to a further embodiment of the invention, the effectiveness ofthe monoclonal antibody or a fragment thereof in alleviating thesymptoms, preventing or treating disease may be improved byadministering an antibody or fragment thereof according to the inventionserially or in combination with another agent that is effective for thesame clinical objective, such as another antibody or a fragment thereofdirected against a different epitope than that of the antibody accordingto the invention, or one or more conventional therapeutic agents knownfor the intended therapeutic indication, e.g. arthritis includingosteoarthritis and rheumatoid arthritis. Other diseases are tendinitis,e.g. peritendinitis, tenosynovitis, insertitis, tendinous bursitis andapophysitis, and atherosclerosis.

Suitable pharmaceutically acceptable agents affecting such indicationsmay be anti-inflammatory drugs such as non steroidal anti-inflammatorydrugs (NSAIDS) for the treatment of joint diseases e.g. osteoarthritis,rheumatoid arthritis; anti-cytokine agents e.g. anti-TNF antibodies,interleukin receptor antagonist, matrix metalloproteinase (MMP)inhibitors or bone morphogenic proteins (BMP); local anaesthetics foruse post-operatively following orthopaedic surgery for the treatment ofpain management or hypolipidemic drugs for treatment of atheroscleroticplaque, matrix metalloproteinase (MMP's) inhibitors or bone morphogenicproteins (BMP's).

Combination of Possible Drugs with Ab for Delivery

In another embodiment of the invention, a monoclonal antibody binding tothe extracellular part of the I-domain of integrin alpha10beta1 or afragment thereof, such as e.g. the antibody 365 or a fragment thereof,or a pharmaceutical composition thereof, will be used as a vehicle toenable the targeted the delivery of other known therapeutic agents toalpha10beta1 expressing cells. Such cells include chondrocytes, MSCs,macrophages, monocytes, synovial cells, tenocytes, myoblasts,osteoblasts, and fibroblasts.

The expression of the exogenous genetic material in vivo, is oftenreferred to as “gene therapy”. Disease states and procedures for whichsuch treatments have application include genetic disorders and diseasesof joints. Cell delivery of the transformed cells may be effected usingvarious methods and includes infusion and direct depot injection intojoints, periosteal, bone marrow and subcutaneous sites.

In one embodiment, the pharmaceutical composition is administered as anadministration vehicle, comprising said monoclonal antibody or afragment thereof combination with other gene or bio delivery systems.The combined administration vehicle comprising said monoclonal antibodyor a fragment thereof is used in combination with other gene or biodelivery systems to selectively target integrin alpha10beta1 expressingcells.

Such a vehicle would involve coupling the antibody or a fragment thereofto a delivery vehicle which would include, for example, virus,liposomes, microcapsules, nano-capsules, plasters, sublingual tablets,and polymer matrices such as poly(orthoesters),polylactide:polyglycolide polymers, and coupling the treatment agenteither to the antibody or a fragment thereof, or to the deliveryvehicle. Examples of agents that could be coupled are non-steroidalanti-inflammatory drugs (NSAIDS), local anaesthetics, cytokineantagonists such interleukins-1 receptor antagonist, type II solublereceptor of interleukins-1, anti-TNF-α monoclonal antibodies, solubleTNF-α receptor, anti-inflammatory cytokines such as IL-4, IL-10, IL-11,growth factors such as fibroblast growth factor, insulin growth factor,transforming growth factor-beta, hepatocyte growth factor,platelet-derived growth factor, parathyroid, hormone-related peptide,bone morphogenic proteins, Indian hedgehog, sonic hedgehog, SOX proteinssuch as SOX5-6, and SOX9, BMP's such as BMP 2 and 7, or inhibitors ofmetalloproteinases.

In a further embodiment, the antibody or a fragment thereof will be usedas a vehicle to enable targeted gene-delivery of agents to alpha10beta 1expressing cells. Cells to be targeted for gene-delivery include thosecells of the skeletal system comprising, cartilage, bone, tendon,ligament and muscle, or cells in an atherosclerotic plaque.

One drawback of the currently available vectors for gene therapy is thelack of a specific cell surface target on cells such as chondrocytes,MSCs and ES cells in gene delivery. It is therefore of great advantageto be able to target the cell of interest e.g. a chondrocyte, by use ofan antibody or a fragment thereof such as an antibody, or a fragmentthereof, of the present invention, e.g. the antibody 365.

In one embodiment the antibody or a fragment thereof may be used inconjunction with a viral or non-viral delivery system for the in vivodelivery of a gene or a part thereof directly to the target tissue orcell of interest, e.g. alpha10 expressing cells of cartilage.

In another embodiment a gene is delivered into a alpha 10beta1expressing cell, preferably MSCs or chondrocyte using a virus, viralvectors include retroviruses, adenoviruses, adeno-associated viruses(AAV), herpes simplex virus and lentivirus.

Especially genes may be transferred to chondrocytes via the integrinalpha10beta1 using adenovirus and the monoclonal antibodies according tothe inventions, such as the mAb365 antibody. This may be done asdescribed in Barry et al 2003 and Parrott et al 2003.

In one embodiment a gene or a combination of genes are delivered into aMSCs or chondrocyte by a non-viral method. Non-viral delivery systemsinclude the use of naked DNA, cationic liposomes, cationic lipids andpolymers as well as DNA/cationic liposome/polycation complexes.

Suitable Genes of Interest to be Delivered

Examples of suitable genes to be delivered include growth factors suchas insulin-like growth factor-1 (IGF-1), transforming growth factor-beta(TGF-β), fibroblast growth factors, and bone morphogenic proteins,transcription factors such as SOX-9, SOX-5, SOX-6, certain signallingmolecules such as SMADs and molecules that inhibit apoptosis such asBCL-2, enzyme inhibitors such as metalloproteinase inhibitors, promotersfor genes of extracellular matrix molecules such as collagens e.g.collagen type II.

Methods are applicable to rodents including mice, rats, rabbits, as wellas humans.

In another embodiment cells expressing alpha10beta1 isolated using theantibody according to the invention or a fragment thereof, e.g. theantibody 365 or a fragment thereof, such as chondrocytes, are for use inautologous chondrocyte transplantation. The cells are then geneticallymodified while undergoing expansion in culture. Viral vectors such asretrovirus, adenovirus, AAV, and lentivirus can readily transduce thesecells. The antibody according to the invention or a fragment thereof inconjunction with a viral delivery system may be used to targetchondrocytic cells expressing alpha10beta1.

In yet another embodiment mesenchymal stem cells isolated with themonoclonal antibody according to the invention or a fragment thereof,for use in tissue repair are genetically modified using viral vectorssuch as retrovirus, adenovirus, AAV, and lentivirus and other viralvectors known to the skilled man in the art. Antibody 365 in conjunctionwith a viral delivery system can used to target MSCs expressingalpha10beta1.

In one embodiment the antibody or a fragment thereof may be used inconjunction with a viral or non-viral delivery system for the in vivotransfer of a gene(s) directly to the damaged tissue, e.g. of cartilage,tendon, bone, ligament, muscle etc. The antibody 365 or a fragmentthereof and the gene(s) of interest may be delivered locally to the siteof tissue damage.

In another embodiment the chondrocytes isolated with an antibodyaccording to the invention or a fragment thereof for use in autologouschondrocyte transplantation are genetically modified while undergoingexpansion in culture, i.e. ex vivo gene transfer. An antibody or afragment thereof is then used in conjunction with a viral/non-viraldelivery system and can used to target those cells that have notde-differentiated and thus lost their chondrocytic phenotype. Thesecells are then injected intraarticularly back into the joint of thepatient from which they were harvested.

In still another embodiment, MSCs with an antibody according to theinvention or a fragment thereof coupled to a gene of interest ormodified using viral/non-viral vectors can be transplanted together witha suitable tissue scaffold or matrix. Suitable tissue scaffolds aredescribed above.

In yet another embodiment, MSCs with an antibody according to theinvention or a fragment thereof coupled to the gene of interest can betransplanted directly into the damaged tissue, e.g. damaged tissuesincluding those mentioned previously.

In yet another embodiment, MSCs with an antibody according to theinvention or a fragment thereof coupled to the gene of interest can betransplanted together with a suitable tissue scaffold or matrix.Suitable scaffolds are mentioned above.

In still another embodiment, non-viral methods using an antibodyaccording to the invention or a fragment thereof for gene transfer areused. Such methods may be based on e.g. cationic lipids, or polyplexconjugates.

Various types of synthetic vectors have been developed for genetransfer, such as cationic-lipid, and polymer-based systems.Cationic-lipid/DNA complexes, i.e. lipoplexes, may be used in which anantibody according to the invention or a fragment thereof may becomplexed with the liposome containing the DNA of the gene of interest.Such genes are as mentioned previously, and include e.g. growth factors.

In one embodiment an antibody according to the invention or a fragmentthereof is incorporated into liposomes together with DNA of a gene ofinterest and injected locally into a joint in the form of polyplex, ormolecular, conjugates.

Mammals in the Need Thereof

According to the invention, a mammal in the need thereof may be a humanbeing in the need thereof. Examples of a human being in the need thereofis a human being with a bone or joint disease, e.g. arthritis includingosteoarthritis and rheumatoid arthritis, osteoporosis or rachitis. Otherdiseases are tendinitis, e.g. peritendinitis, tenosynovitis, insertitis,tendinous bursitis and apophysitis and atherosclerosis.

Further, a mammal in the need thereof may be a any mammal such as ahorse, a cow, a pig or piglet, dog, or primate.

Further, a mammal in the need thereof may be a rodent, including allmembers of the phylogenetic Rodentia, such as a rabbit, a mouse, guineapig, or a rat.

Routes for Administration

The antibody or fragments thereof of the present invention can beadministered to an individual by an appropriate route, either alone orin combination with—before, simultaneously with, or after—another drugor agent. For example, the antibody of the present invention can also beused in combination with other monoclonal or polyclonal antibodies, withexisting products, such as commercially available products used inprophylactic or therapeutic treatments of joint diseases. The antibodyor fragments of the present invention can be used as separatelyadministered compositions given in conjunction with antibiotics and/orantimicrobial agents.

An effective amount of an antibody or fragments thereof is administered.An effective amount is an amount sufficient to achieve the desiredtherapeutic effect, including prophylactic, under the conditions ofadministration, such as an amount sufficient for inhibition orstimulation of alpha10beta1, and thereby, modulate, such as prevent,alleviate, or treat, a joint disease.

A variety of routes of administration are possible including, but notnecessarily limited to, oral, dietary, topical, parenteral, e.g.,intravenous, intraarterial, intramuscular, subcutaneous,intra-articular, or intraperitoneal, depending on the joint disease orcondition to be treated. Other suitable methods of administration canalso include rechargeable or biodegradable devices and slow releasepolymeric devices. The pharmaceutical compositions of this invention canalso be administered as part of a combinatorial therapy with otheragents.

Kit According to the Invention

The invention further discloses a kit, comprising the monoclonalantibody an antibody according to the invention or a fragment thereof ora fragment thereof.

Kits for use in detecting the presence of a mammalian integrinalpha10beta1 in a biological sample can also be prepared. Such kits willinclude an antibody according to the invention or a fragment thereof,such as antibody 365 or fragment thereof which binds to an I-domain of amammalian integrin alpha10beta1, as well as one or more ancillaryreagents suitable for detecting the presence of a complex between theantibody or fragment and integrin alpha10beta1 or portion thereof. Theantibody compositions of the present invention may be provided inlyophilized form, either alone or in combination with additionalantibodies specific for other epitopes.

The antibodies, which may be labelled or unlabelled, may be included inthe kits with adjunct ingredients e.g., buffers, such as Tris, phosphateand carbonate, stabilizers, excipients, biocides and/or inert proteins,e.g., bovine serum albumin. For example, the antibodies can be providedas a lyophilized mixture with the adjunct ingredients, or the adjunctingredients can be separately provided for combination by the user.Generally these adjunct materials will be present in less than about 5%weight based on the amount of active antibody, and usually will bepresent in a total amount of at least about 0.001% weight based onantibody concentration.

Where a second antibody capable of binding to the monoclonal antibody isemployed, such antibody can be provided in the kit, for instance in aseparate vial or container. The second antibody, if present, istypically labelled, and may be formulated in an analogous manner withthe antibody formulations described above.

The kit includes, in an amount sufficient for at least one isolation, anmonoclonal antibody of the present invention or a fragment thereof as aseparately packaged reagent, or in one further embodiment as a reagentin combination with a solid phase support or bead. Instructions for useof the packaged reagent are also typically included.

In one embodiment the present invention relates to a kit for isolatingES cells, MSCs or chondrocytes from a human subject.

In a further embodiment, the monoclonal antibody or a fragment thereofcomprises a detectable label.

In one further embodiment the kit comprises an antibody according to theinvention or a fragment thereof and an anti-immunoglobulin labelledantibody or a fragment thereof, for example PE-labelled goat anti-mouseIgG, suitable for use in FACS analysis.

In one further embodiment the kit comprises an antibody according to theinvention or a fragment thereof coupled to solid phase support or bead.Examples of solid supports of beads are given in the paragraphs above.

In one further embodiment, an antibody according to the invention isprovided in a solution.

In one further embodiment, an antibody according to the invention isprovided lyophilized to be dissolved upon usage.

Further, a kit for production of an antibody according to the inventionor a fragment thereof, such as the antibody 365 or a fragment thereof,is disclosed, comprising a hybridoma cell line, such as the hybridomacell line mAb365 according to the invention.

In one embodiment the kit for production of an antibody according to theinvention or a fragment thereof, such as the antibody 365 or a fragmentthereof, a cell culture medium for said hybridoma cell line is included.

While the invention has been described in relation to certainembodiments the skilled person may foresee other not mentionedembodiments, variations or combinations, that are still within the scopeof the claims.

By the expression “comprising” as used herein we understand includingbut not limited to the stated items.

The invention will now be described by the following non-limitingexamples.

EXAMPLES Example 1 Generation of Clone 365 Objective

The objective with this example was to generate a monoclonal antibodyagainst the I-domain of the extracellular domain of alpha10.

Materials and Methods

The Antigen

For the production of a monoclonal antibody specific for alpha10integrin, alpha10 knockout mice were immunized with recombinant alpha10I-domain purified from an alpha10 I-domain-expressing cell line. Thecell line was generated by transfecting HEK 293-EBNA cells with theexpression vector pCEP4 coding for His-tagged alpha10 I-domain alone orfused to alkaline phosphatase (AP).

The recombinant proteins have been designed so that they were secretedinto the culture medium from where they were affinity purified on NiNTAagarose (Qiagen). Purity was confirmed by electrophoresis.

Immunisation

Mice were immunized intramuscularly with 2-10 μg alpha10I-domain-alkaline phosphatase fusion protein mixed with the mouseadjuvant Immuneasy (Qiagen). Fifteen days later the mice were boostedwith the same antigen. A further 2 or 3 boosts with alpha10 I-domain (4μg) administered subcutaneously at the base of tail at 2-week intervals,was required to reach the desired specificity response in both ELISA andFACS.

Two days after the last immunization, spleen cells from the mice werefused with NSO myeloma cells using polyethylene glycol. Fused cells wereseeded in a 96-well microplate and grown in DMEM/F12 (Invitrogen) mediumcontaining BM Condimed H1 (Roche) and HAT (hypoxanthine, aminopterin,thymidine mixture Sigma) selection.

Hybridoma cell clone supernatants were tested for anti-alpha10 antibodyproduction by their ability to bind to immobilized alpha10 I-domain byELISA and by binding to a cell line expressing alpha10beta1 in FACSanalysis.

A total of 29 alpha10 I-domain positive clones were identified by ELISA,and one of them named the antibody 365 was found to bind specifically toalpha10beta1 by FACS. Positive hybridoma cell lines were subcloned threetimes by limiting dilution techniques.

Isotyping of the antibody secreted by clone 365 by Isostrip, a mousemonoclonal antibody isotyping kit by Roche (Switzerland), identified theantibody to be an IgG2aκ

Results

One hybridoma clone, 365, was stable after subcloning. The monoclonalantibody produced by the hybridoma is further characterised below.

Example 2 Immunoprecipitation of Integrin Alpha10Beta1 with the Antibody365 Objective

The objective with this example was to demonstrate the specificity ofthe antibody 365 for the whole integrin (alpha10beta1) byimmunoprecipitation (IP).

Materials and Methods

In the following experiment, polyclonal antibodies against thecytoplasmic domains of integrin subunits alpha10 and alpha11 were usedas control antibodies. These polyclonal antibodies had previously beenshown to specifically immunoprecipitate integrins alpha10beta1 and.alpha11beta1 respectively from cell lysates.

C2C12 cells transfected with integrin subunit alpha10 or alpha11(negative control) were grown in DMEM medium with 10% FCS. Cellsadherent on the plate were washed once with PBS and then surfacebiotinylated using 0.5 mg/ml Sulfo-NHS-LC-biotin (Pierce) in 4 ml PBSfor 20 min on ice. Cells were then washed once with PBS and 10 ml 0.1Mglycine/PBS were added for 5 min on ice. After washing once with PBScells were lysed in 1 ml lysis buffer (1% NP-40, 10% glycerol, 20 mMTris/HCl, 150 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, protease inhibitorcocktail Roche, pH7.5) on ice. The cell lysate was collected with aplastic scraper and spun down at 15.000 g for 10 min. The supernatantwas removed and incubated with 1 μl of α10 pre-immune serum and then 20μl Prot G Sepharose (Amersham) in 100 μl lysis buffer were added. Afterrotating 1 h at 4° C. the lysate was centrifuged for 1 min at 8000 rpmand the supernatant removed. For each immunoprecipitation 150 μl celllysate supernatant were pipetted into an eppendorf tube and 1 μl ofantiserum or monoclonal antibody solution was added. The antibodies usedwere mouse the antibody 365, rabbit-anti-human α10 serum andrabbit-anti-human α11 serum, respectively (both sera against thecytoplasmic domains of the integrins). After 2 h rotating at 4° C., 20μl prot G Sepharose (Amersham) in 100 μl lysis buffer were added and themixture further rotated for another 45 min. The Sepharose-beads werethen spun down briefly and washed three times with lysis buffer. 20 μlSDS PAGE sample buffer (including 100 mM DTT) were added to theSepharose beads and the samples were boiled for 5 min. 5 μl of eachsample were run on an 8% straight gel (Novex) and thenelectro-transferred onto a PVDF membrane. The membrane was blocked in 2%BSA/TBST for 1 h, washed once with TBST and then incubated with 2 μlExtravidin-peroxidase (Sigma) in 8 ml blocking buffer. After 1 h theExtravidin-peroxidase solution was removed and the membrane washed 3×20min in TBST. Surface biotinylated proteins were then detected with ECL(Amersham) and visualised on a photographic film.

Results

The results in FIG. 2 demonstrate that the antibody 365 is able toimmunoprecipitate the whole integrin alpha10beta1 (lane 3); cytoplasmicpolyclonal alpha10 antibody was used as a positive control to confirmthe presence of integrin alpha10beta1 on the surface of thealpha10-transfected C2C12 cells (lane 1). The antibody 365 was specificfor the alpha10beta1 integrin since it did not immunoprecipitateintegrin alpha11beta1 from alpha11-transfected C2C12 cells (lane 6) orany other protein. Polyclonal serum against the cytoplasmic domain ofintegrin alpha11 subunit (lane 5) was used a positive control for alpha11 transfected cells.

Example 3 ELISA Objective

The objective with this example was to demonstrate the specificity ofthe antibody 365 for the I-domain of the integrin alpha10 chain by ELISA(enzyme linked immunosorbent assay).

Materials and Methods

Soluble recombinant I-domain (10 μg) of alpha10, alpha11, alpha1 orcontrol protein (alkaline phosphatase) was coated in a 96 well ELISAplate (Maxisorp Nunc) overnight in PBS.

Hybridoma culture supernatant containing approximately 1 μg/ml of theantibody 365 was applied and specific binding of the antibody to alpha10I domain was detected by horseradish peroxidase-conjugated goatanti-mouse IgG and subsequently peroxidase substrate (OPD SigmaFast,Sigma). The absorbance of the calorimetric change was determined at 492nm.

Results

The results in FIG. 3 confirm that the antibody 365 specificallyrecognises the I-domain of the integrin subunit alpha10. No reactivitywas observed with control (AP) or the I-domains of the integrin alpha1and alpha11.

Example 4 Cell Adhesion Assay Objective

The objective of example 4 is to show that the antibody 365 can modulatethe binding of alpha10beta1 to collagen type H.

Materials and Methods

48-well plates (Nunc) were coated with collagen type II or BSA (10 μg/ml150 μl/well) in PBS 4° C. overnight, followed by blocking with 2% BSA inPBS for 1 h at room temperature. Cells were trypsinized, washed and thenseeded on collagen or BSA coated wells at specific ion-concentrations inthe presence or absence of antibodies Cells were seeded at 50,000cells/well, and were allowed to attach for 1 h 37° C. Wells were washedtwo times with PBS. Cell numbers of adherent cells were determined usingthe hexosaminidase test as follows: —Attached cells were lysed in 150 μlsubstrate solution (7.5 mM p-Nitrophenyl-N-Acetyl-β-D-Glucosamine, 0.05Msodium acetate pH 5, 0.25% Triton X-100). The plates were incubated at37° C. for 2.5 h. 60 μl of the cell lysate were transferred to amicrotiter plate (Nunc) and mixed with 90 μl developing buffer (5 mMEDTA, 50 mM Glycine pH 10.4)

The absorbance at 405 nm was read and used as a measure of cell number.For each cell line used, a cell number standard was made. Eachexperiment was performed in triplicates.

Results

In FIG. 4 a is shown that the antibody 365 inhibits binding ofalpha10beta1-expressing C2C12 cells to collagen II in the presence of 1mM Mg²⁺ and 1 mM Ca²⁺. Control (no Ab) and 1B4 (isotype control) showedno inhibition of binding.

In FIG. 4 b is shown that binding of alpha11beta1-expressing C2C12 cellsto type II collagen is not inhibited by the antibody 365. Control (noAb) and 1B4 (isotype control) showed no inhibition of binding.

Example 5 Identification of Cells Expressing Alpha10 Integrin by FACSObjective

The objective with this example is to use the antibody 365 to identifycells expressing human alpha10beta1 integrin.

Materials and Methods

Alpha10 and alpha11-transfected C2C12 and non-transfected C2C12 weretrypsinized, washed with PBS and then incubated for 20 min with theantibody 365 1 μg/ml in PBS supplemented with 1% BSA. Labelled cellswere washed twice with PBS/1% BSA and then incubated for 20 min with PElabelled goat-anti-mouse Ig (Pharmingen, BD Biosciences) at aconcentration of 1 μg/ml in PBS/1% BSA. Cells were thereafter washedtwice in PBS/1% BSA and were analysed on a FACSort® (Becton-Dickinson)by collecting 10,000 events with the Cell Quest® software program(Becton-Dickinson).

Results

FIG. 5 shows the identification of cells expressing alpha10 using theantibody 365 in FACS analysis. In the FACS assay, the antibody 365 boundto C2C12 cells transfected with human alpha10 integrin-subunit, shown inthe upper middle panel. This was seen as a displacement in the FACShistogram to the right. The antibody the antibody 365 did not bind toC2C12 cells transfected with human alpha11 integrin-subunit, as shown inthe upper right panel, or untransfected C2C12 cells, as shown in theupper left panel. The lower panels represent secondary antibody, alonewhich did not bind to any of the cells tested.

Example 6 Selection of Cells Binding to the Antibody 365 by MACS®Objective

The objective with this example is to positively select cells expressingalpha10beta1 by MACS® beads.

Materials and Methods

A mixed cell population containing alpha10beta1 expressing andnon-expressing HEK 293-EBNA cells was subjected to positive selectionfor alpha10-expressing cells by using magnetic bead separation, MACS

Cells were trypsinized, washed in PBS and then incubated with theantibody 365 at 1 μg/ml in PBS supplemented with 2 mM EDTA and 0.5% BSA(MACS buffer) for 15 min on ice. Incubated cells were thereafter washedtwice with PBS and then resuspended in 80 μl MACS buffer and 20 μlgoat-anti-mouse IgG Microbeads (Miltenyi Biotec Germany). After beenincubated for 15 min on ice, the labelled cells were washed twice inMACS buffer and resuspended in 500 μl MACS buffer. The suspension werepassed over a LS separation column containing a magnet (Miltenyi Biotec,Germany) and the column was washed with 3 ml MACS buffer three times toremove non-labelled cells.

The column was removed from the magnet and the labelled cells wereeluted with MACS buffer and collected by centrifugation.

The three different cell fractions (cells before selection, flow throughand positively selected cells) were incubated for 20 min with theantibody 365 1 μg/ml in PBS supplemented with 1% BSA. The cells werethen washed twice with PBS/1% BSA and then incubated for 20 min with PElabelled goat-anti-mouse Ig (Pharmingen, BD Biosciences) at aconcentration of 1 gμ/ml in PBS/1% BSA. Cells were thereafter washedtwice in PBS/1% BSA and then analysed on a FACSort® (Becton-Dickinson).

Results

The effectiveness of the selection was determined by flow cytometryanalysis, FACS, and is shown in FIG. 6. Cells before selection, flowthrough and eluted cells were stained with the antibody 365. The alpha10positive populations are shifted to the right as displayed in thehistograms. Out of 13 million of cells in the starting population, 1.48million i.e. equivalent to 11% were positively selected. The positivelyselected cell fraction contained almost no alpha10beta1-negative cells.The flow-through fraction contained almost no alpha10-positive cells,confirming that the MACS-separation had efficiently removedalpha10-positive cells from the mixed cell population that had been usedas starting material.

Example 7 Identification of a Population of hMNC Binding to the Antibody365 Objective

The objective of this example is to identify a sub-population of humanmononuclear cells using the antibody 365.

Materials and Methods

Human mononuclear cells (hMNC) were isolated from the bone marrow of theiliac crest of normal adults. About 20 to 30 ml of marrow aspirate wascollected into a syringe containing 6000 units of heparin to preventclotting. The marrow sample was diluted 1:1 with Iscove's modifiedDulbecco's medium (IMDM)+5% FCS. The bone marrow suspension was thefiltered through a 50 μm pore size mesh and 15 ml of Lymphoprep (Roche)added into 50 ml tubes. Bone marrow cells (25 ml) were carefully layeredon the top of the Lymphoprep layer, avoiding mixing. The cells were thencentrifuged for 30 min at room temperature at 400×g. The cells from theinterface were then transferred into a 50 ml tube containing 25 ml ofIMDM+5% FCS before centrifugation at 500×g for 15 min at 4° C. Thesupernatant was removed and 5.0 ml of buffer added (sterile PBS withoutCa²⁺ and Mg²⁺ supplemented with 2 mM EDTA containing 5% FCS).

Identification of a Population of hMNCs by Flow Cytometry

Purified mononuclear cells from above were divided in two tubes andincubated for 20 min on ice with or without the antibody 365 (1 μg/ml)in PBS containing 1% foetal calf serum (FCS).

Labelled cells were washed once with PBS/1% FCS and then incubated for20 min on ice with PE labelled goat-anti-mouse IgG (1 μg/ml PharmingenBD Biosciences) this was followed by another wash with PBS/1% FCS. Thecells were then incubated 20 min on ice with FITC labelledmouse-anti-human CD45 (1.2 μg/ml, BD Biosciences) for the identificationof lymphocytes in the mononuclear preparation, and followed by a washwith PBS/1% FCS

Labelled cells were analysed on a FACSCalibur® (Becton-Dickinson) bycollecting a total of 1,000,000 events with the Cell Quest® softwareprogram (Becton-Dickinson).

Results

FIG. 7 shows identification of a population of integrinalpha10-expressing hMNCs using the antibody 365 in MACS analysis (lowerpanel). The upper panel shows MACS analysis in the absence of theantibody 365.

Example 8 Immunohistochemistry Objective

The objective with this example is to show the binding in situ of theantibody 365 to human articular cartilage.

Materials and Methods

Tissue sections were warmed for 30 min at room temperature before thetissue was surrounded with PAP pen (Histolab) and fixed in acetone(Merck) for 10 min at −20° C. The tissue was then washed in PBS(Gibco/Invitrogen) at room temperature for 15 min, with one change ofPBS, followed by digestion in 2 mg/ml hyaluronidase (Sigma EC 3.2.1.35)at 37° C. for 30 min. The digested tissue was washed twice in PBS undera 15 min incubation before blocking for 30 min at room temperature with2% donkey serum (Jackson ImmunoResearch Laboratories, Inc.) in PBS.Primary antibody the antibody 365 was diluted 1:400 in 2% donkey serumin PBS and samples incubated for 75 min at room temperature. Sampleswere washed twice in PBS at room temperature during a 15 min incubationbefore addition of secondary antibody donkey-anti-mouse Cy3 (JacksonImmunoResearch Laboratories, Inc.) for 60 min at room temperature.

Samples were washed twice in PBS at room temperature during a 15 minincubation and slides were mounted with Vectashield Mounting Medium(Vector Laboratories). Tissue sections were viewed using a microscopeequipped with a Cy3 filter.

Results

FIG. 8 shows immunolocalisation of integrin alpha10beta in humanarticular cartilage using the antibody 365 (upper panel). The secondaryantibody only did not bind to the chondrocytes (lower panel).

Example 9 Use of mAb365 for Identification of a Population ofAlpha10⁺-Chondrocytes Objective

The objective with this example is to identify a population ofalpha10⁺-chondrocytes.

Materials and Methods

Human chondrocytes were isolated from human normal cartilage bycollagenase digestion (see protocol below).

Extraction of Human Chondrocytes

Extraction of human chondrocytes from human articular cartilage isperformed in the following manner. The protocol is described byBrittberg et al in (1994) N. Engl. J. Med 331:889-895.

-   1. Upon receiving the human cartilage, wash 3× in PBS (−/−)+1:100    Penicillin/Streptomycin (PEST)+1:250 fungizone.-   2. Place cartilage that is not to be dissected into culture media    (DMEM at 37° C. in a 15 cm dish.-   3. Dice the cartilage into 1-2 mm³ pieces.-   4. Weigh out pronase (7001 U/ml=10 mg/ml), dissolve in medium    (+1:100 PEST+1:250 fungizone) without serum, sterile filter and    prewarm solution to 37° C. before using.-   5. Incubate in pronase for no more than 30 mins at 37° C. with very    gentle rolling.-   6. Allow pieces to settle, remove the supernatant and discard.-   7. Wash pieces three times in PBS−/− (+1:100 PEST+1:250 fungizone)    to remove all pronase.-   8. Weigh out collagenase (3501 U/ml) and dissolve in media without    serum (+1:100 PEST+1:250 fungizone), sterile filter, and prewarm    (37° C.) solution.-   9. Digest the cartilage at 37° C. overnight with rolling.-   10. Allow any remaining pieces to settle and then pipette    supernatant through a 70 μm cell strainer and centrifuge at 1500 rpm    for 10 mins.-   11. Check the supernatant for cells after spinning and respin if    necessary. If there are many pieces left, redigest in new    collagenase (as above) at 37° C. with rolling.-   12. To remove all collagenase, wash the cells three times in PBS    (−/−)+PEST (1:100)+fungizone (1:250)+10% serum) (resuspend cells in    PBS, then spin at 1500 for 10 minutes).-   13. Resuspend in medium containing 10% serum and count—use Trypan    blue exclusion.-   14. Spin down and plate out at the required density or prepare for    freezing.

Plating of Cells

The isolated cells were plated out using the following method:

1. Resuspend cells after counting in serum-free medium and plate out for2-3 hrs.

2. Remove medium from cells and add medium containing 10% serum

3. Plating density: -T75 high density 8-10×10⁶ cells/12 ml: low density5-6×10⁶ cells/12 ml.

4. Change medium every 2-3 days, depending on density of cells.

Cells were cultured in DMEM/F12 medium supplemented with 10% FCS, PESTand ascorbic acid 50 μg/ml. Cells were detached by day 1, or after 1, 2and 6 weeks with trypsin/EDTA, and washed in PBS containing 1% BSA bycentrifugation at 1200 rpm for 5 minutes.

FACS-Analysis of Cultured Cells

Cells were stained with mAb365 (1 μg/ml), or isotype control IgG. At theend of the incubation cells were washed again as described above, andthe bound antibodies were detected by incubating cells with PEconjugated goat-anti mouse antibody (Pharmingen) for 20 minutes followedby another wash as above.

Cells were resuspended and analyzed by flow cytometry with FACSort(Becton Dickinson FACS system).

Results

The results in FIG. 9 show a summary of the FACS data collected afteranalysis and indicate the percentage alpha10 positive cells identifiedby the mAb365 antibody in FACS analysis.

FACS analysis of cells was performed on cells that were detached on day1, or after 1, 2 and 6 weeks. Cells were detected by incubating with PEconjugated goat-anti mouse antibody. Under the period of culture, thepercentage alpha10 positive cells decreased from approx 70% at day 1 to10% after 6 weeks in culture.

Example 10 Isolation of Alpha10-Positive Chondrocytes Using MagneticCell Sorting with mAb365 Objective

The objective of this example is to show that mAb365 can be used forsolid phase cell sorting, for separation of alpha10-positive cells.

Materials and Methods

The human chondrocyte-fraction isolated in Example 9 was furtherfractionated into alpha10 positive and alpha10 negative cells by usingmagnetic cell sorting.

The human chondrocytes were labelled with 10 μg/ml mAb365 (anti-I-domainof alpha10 integrin receptor) for 20 minutes at 4° C., washed andlabelled with goat anti-mouse IgG micro beads (Miltenyi Biotec, Germany)for 20 minutes in 4° C.

Alpha10 positive cells were isolated by positive selection with an LSmidiMACS column (Miltenyi Biotec, Germany). This procedure is performedaccording to the manufacturers' instructions.

Remaining positive cells in the alpha10 negative fraction, from afterthe positive selection were depleted with an LD depletion column(Miltenyi Biotec, Germany).

1.6×10⁵ cells of the positive and negative fraction were used for mRNAisolation. These cells were then used for a cDNA synthesis.

The cDNA was used for Quantitative PCR, which was performed on a LightCycler (Roche) using FastStart DNA Master SYBR Green I using thefollowing conditions: Denaturation for 10 min at 95° C., followed by 40cycles with an annealing temperature of 65° C. with specific primers foreach transcript (GAPDH, collagen I and collagen II). All PCR data werenormalised against GAPDH.

Collagen Primers used:

Human Collagen I COL I forward 3′-5′ gCTTCCCTggTCTTCCTg COL I reverse3′-5′ TCTCACCACggTCACCCT Human Collagen II COL II forward 3′-5′CAggggTgAACgAggTTT COL II reverse 3′-5′ gAggTCCAACTTCTCCCTTCT

Measurement of Collagen Type II Production

For measurements of collagen content, either the total amount ofcollagen can be determined using the hydroxyproline assay (Woessner J. F1976 In: The Methodology of Connective Tissue Research. Ed: Hall D pp227-233) or collagen synthesis can be measured by radiolabelling with ³HProline (Scutt et al (1992) Anal. Biochem 203:290-294).

As an example, measurement of hydroxyproline content is performed in thefollowing manner: Samples containing collagen (typically collagen typeII) are hydrolysed in 6.0 M HCl for 16 hours at 110° C. to liberatehydroxyproline.

After neutralization each sample is diluted at least 15 times to preventthe salt concentration from influencing the assay.

The samples are then dried under vacuum.

Method:

a. Samples (1-5 μg of hydroxyproline) are made up to 2.0 ml with assaybuffer.b. Add 1.0 ml of Chloramine-T reagent and stand for 20 minutes at roomtemperature.c. Add 1.0 ml of freshly prepared dimethylaminobenzaldehyde reagent andmix thoroughly.d. Incubate the tubes at 60° C. for 15 minutes and cool in tap water for5 minutes.e. Measure the absorbance at 550 nm within 45 minutes.Note: The hydrolysate may be passed over short columns of Dowex-50-x-8(H+ form, 200-400 mesh) to remove coloured material and impurities ifnecessary.

Reagents:

1. Stock buffer contains 50 g of citric acid (H₂O), 12 ml of glacialacetic acid, 120 g of sodium acetate, 3H₂O and 34 g of NaOH in 1.0 litreof solution. A few drops of toluene are added as preservative.2. Assay buffer: The stock buffer solution is diluted tenfold with H₂O.3. Chloramine-T reagent. 1.41 g of chloramine-T is dissolved in 20.7 mlof H₂O and mixed with 26 ml of n-propanol and 53.3 ml of stock buffer.(This reagent is stable at 4° C. for 2 weeks)4. Dimethylaminobenzaldehyde reagent. 15 g ofp-dimethylaminobenzaldehyde is suspended in 60 ml of n-propanol and 26ml of perchloric acid (60%) is added slowly (N.B. Use a fume hood withprotective goggles). This reagent must be freshly prepared.

Results

The results in FIG. 10 demonstrate the levels of collagen type II andcollagen type I RNA in human chondrocytes separated by magnetic cellseparation using mAb365 upon the basis of expression of the integrinalpha10beta1. The results show that:

-   -   i) The expression of collagen type II is greater than the        expression of collagen type I in those cells that express the        integrin alpha10beta1 i.e. are alpha10 positive.    -   ii) The expression of collagen type I is greater than the        expression of collagen type II in those cells that do not        express the integrin alpha10beta1 i.e. are alpha10 negative.

Discussion

This result indicates that alpha10-expressing cells produce mRNA codingfor an extracellular matrix component, namely collagen type II, that isconducive to a cartilage-like matrix.

Conversely, cells lacking alpha10 produce more collagen type I mRNA, anextracellular matrix component associated with a more‘fibrocartilage-like’ matrix.

Example 11 Identification of Murine Alpha10 by mAb365 Objective

The objective of this example is to test whether it identifies murinealpha10 expressed on chondrocytes.

Materials and Methods

Chondrocytes were isolated from rib cartilage from newborn wild-type oralpha10 knockout mutant mice using the following protocol described byBengtsson et al. (Matrix Biology 2001 20 (8):565-76)).

-   -   1. Dissect out the entire ribcage from the mouse.    -   2. Place the tissue in DMEM-PS (DMEM+1:50 PEST) in a Petri dish        on ice.    -   3. Move the ribs (from ˜3 mice/dish) to a small Petri dish with        3 ml DMEM-PS containing 2 mg/ml collagenase+2% FCS.    -   4. Incubate at 37° C. for 30 minutes, one dish at a time.    -   5. Remove the perichondrium from ribs and move the ribs to a new        small Petri dish with DMEM-PS+2 mg/ml collagenase+2% FCS.    -   6. Incubate at 37° C. until the cells are resuspended.    -   7. Put the cell suspension through a 70 μm cell strainer into a        50 ml Falcon.    -   8. Inactivate collagenase with DMEM-PS+20% FCS.    -   9. Spin 2000 rpm for 10 minutes.    -   10. Wash again with 10 ml DMEM-PS+20% FCS; count cells while        spinning.    -   11. Freeze in cell culture freezing medium at about 1×10⁶        cells/tube. Store at −80° C.

Chondrocytes were grown overnight in culture medium DMEM/F12supplemented with 10% FCS, PEST and ascorbic acid 50 μg/ml before FACSanalysis.

Cells were detached with trypsin/EDTA, and washed in PBS containing 1%BSA by centrifugation at 1200 rpm for 5 minutes.

Cells were incubated with mAb365, isotype control IgG at a concentrationof 1 μg/ml, or FITC conjugated anti-CD29, an antibody that recognisesthe mouse beta 1 integrin.

At the end of the incubation cells were washed again as described above,and further incubated with isotype control or mAb365 and counterstainedwith PE conjugated goat-anti mouse antibody (Pharmingen) for 20 minutesfollowed by a subsequent wash.

Cells were resuspended and analyzed by flow cytometry with FACSort(Becton Dickinson FACS system)

Results and Discussion

FIG. 11 shows the identification of integrin alpha10-expressing mousechondrocytes using the antibody mAb365 in FACS analysis (upper figurepanel).

The lower panel shows a control FACS analysis performed on mousechondrocytes isolated from a integrin alpha10 knockout mouse (describedin WO 03/101497 as well as in Bengtsson et al. Matrix Biology 2001 20(8):565-76).

The results show that mAb365 binds to murine alpha10 as well.

Example 12 Presence of Alpha10 on Human Mesenchymal Stem Cells Objective

The objective with this example is to show the presence of alpha10 onhuman mesenchymal stem cells using mAb365.

Materials and Methods

Human mesenchymal stem cells may be obtained from Poietics oralternatively one may isolate them from bone marrow.

An alpha10+ve population may then be isolated and further differentiatedto see whether they are capable of becoming chondrocytes throughdifferent differentiating conditions as outlined below.

Human mesenchymal stem cells obtained from adult normal bone marrow werepurchased from Poietics/Cambrex (Cat no PT-2501). MAb365 was analysed incombination with other less specific markers for stem cells, such asCD105, CD166, CD44, CD14, CD34, and CD45 (Pieternella et al (2003) J.Haematol 88 (08):845-852; Kirschstein, R and Skirboll, L. R (2001) StemCells: Scientific Progress and Future Directions. NIH Report. Departmentof Health and Human Services).

Purchased cells were thawed and cultured according to Poieticsrecommendation in defined medium (Cat no PT-3001) for 6 days. Cells weredetached with trypsin/EDTA, and washed in PBS containing 1% BSA bycentrifugation at 1200 rpm for 5 minutes.

Cells were typically stained with mAb365 (1-10 μg/ml), isotype controlIgG or P4C10, an antibody recognising the beta1 chain of the integrin.At the end of the incubation cells were washed again as described above,and bound antibodies were detected by incubating cells with PEconjugated goat-anti mouse antibody (Pharmingen) for 20 minutes followedby another wash as above.

Cells were resuspended and analyzed by flow cytometry with FACSort(Becton Dickinson FACS system). The cells were also investigated for thepresence for CD105, CD44, CD14 and CD45 (all antibodies from BDBiosciences/Pharmingen) to determine the mesenchymal stem cellphenotype.

Isolation of MSCs from Human Bone Marrow

As an alternative to using Poietics Mesenchymal Stem Cells, mesenchymalstem cells is isolated from human bone marrow by standard methods(Quirici et al (2002) Exp. Hematol 30 (7):783-791). Bone marrow is takenfrom healthy allogeneic bone marrow transplantation donors, collected inheparinized tubes and layered onto Lymphoprep™ (density 1.077 g/ml,Nycomed, Norway) according to the manufactures' description.

The low-density mononuclear cells (LD-MNC) are then isolated from thehuman bone marrow cells by centrifugation. The LD-MNCs are washed twicein PBS and resuspended in MSCGM (mesenchymal stem cell growth medium)(Poetics, Cambrex Bio Science Walkersville, Inc.).

Mesenchymal Stem Cells is then purified from LD-MNCs by the followingstandard methods: by adhesion to plastic (Pittenger et al (1999) Science184:143), CD45⁻/α-glycophorin A⁻ (Reyes et al (2001) Blood. 98(9):2615-25), CD105⁺ (Conrad et al. (2002). Exp Hematol. 30 (8):887-95)and NGFR⁺ isolation (Quirici et al. (2002) Exp Hematol. 30 (7):783-91).

Isolation of Integrin Alpha10 Positive Cell Population

Integrin alpha10 positive cells is isolated by the following methods:Cells are labelled with a concentration ranging from 1-10 μg/ml mAb365(α10 integrin receptor) for 20 minutes at 4° C., washed and labelledwith goat anti-mouse IgG micro beads (Miltenyi Biotec, Germany) for 20minutes in 4° C. The α10 positive cells are then isolated by positiveselection with an LS midiMACS column (Miltenyi Biotec, Germany). Thisprocedure is performed according to the manufacturers' instructions.Integrin alpha10 negative cells are retained as a control.

Differentiation of Integrin Alpha10 Positive (and Negative) Cells

After identification of an alpha10 positive cell population by any ofthe above methods (and corresponding negative population), it isdesirable to be able to differentiate these cells to a chondrogenicphenotype (and be able to distinguish between the other known phenotypesby e.g. Yoo et al (1998) J. Bone J. Surgery Am 80:1745-1757).

The following methods, known to a man skilled in the art, (Tallheden etal J. Bone. J. Surgery 85A (Suppl2):93-100) may therefore be used todetermine if the alpha10 positive cells identified using the mAb365antibody may be differentiated to a chondrocytes phenotype. Otherdifferentiation conditions will be used as a control. Examples ofdedifferentiation protocols are given below:

Chondrogenic Differentiation

The cells are cultured as pellet mass in DMEM (GibcoBRL, Paisley, UK),insulin transferrin sodium selenite (Sigma, Sweden), 0.1 μMdexamethasone (Sigma, Sweden), 80 μM ascorbic acid-2-phosphate (Sigma,Sweden), 1 mg/ml linoleic acid-bovine serum albumin (Sigma, Sweden), 100U/ml Penicillin, 100 μg/ml Streptomycin (GibcoBRL, Paisley, UK) and 10ng/ml TGF-β3 (R&D Systems Europe Ltd., United Kingdom). To determine thechondrogenic differentiation the pellet cultures are tested for collagentype I and II, aggrecan and versican expression using Q-PCR.

Osteogenic Differentiation

To induce osteogenic differentiation the cells are cultured in DMEM-LG(GibcoBRL, Paisley, UK), 10% FCS (Sigma, St. Louis, Mo.), 50 μM ascorbicacid-2-phosphate (Sigma, Sweden), 0.10 μM dexamethasone (Sigma, Sweden),100 U/ml Penicillin and 100 μg/ml Streptomycin (GibcoBRL, Paisley, UK).At day 11, 2 mM β-glycerophosphate (Sigma, Sweden) is added to theculture. The control cells are cultured without dexamethasone andβ-glycerophosphate. The medium is changed every fourth day, during the21 or 28 days of culture. The mineralization potential of the osteogenicdifferentiated cells are visualised by Von Kossa staining.

Adipogenic Differentiation

To induce adipogenic differentiation the cells are cultured in DMEM-LG(GibcoBRL, Paisley, UK), 10% FCS (Sigma, St. Louis, Mo.), 1 μMdexamethasone (Sigma, Sweden), 60 μM indomethacin (Sigma, Sweden), 0.5mM 3-isobutyl-methyl-xanthine (Sigma, Sweden), 5 μg/ml insulin (Sigma,Sweden), 100 U/ml Penicillin and 100 μg/ml Streptomycin (Gibco,Invitrogen). Every fourth day the cells are cultured during one day inDMEM-LG, 10% FCS (Sigma, St. Louis, Mo.), 100 U/ml Penicillin, 100 μg/mlStreptomycin (GibcoBRL, Paisley, UK) and 5 μg/ml insulin (Sigma,Sweden). The negative control cells are cultured in DMEM-LG (GibcoBRL,Paisley, UK) 10% FCS, 100 U/ml Penicillin and 100 μg/ml streptomycin(GibcoBRL, Paisley, UK). The cells are cultured for 14 days indifferentiation media, the differentiated cells contain lipid vacuolesthat can visualised with Oil Red O staining.

Results

All the cells were positive for CD105, CD166 and CD44, and negative forCD14 and CD45 as determined by flow cytometry, indicating a mesenchymalstem cell phenotype.

Upon investigating the expression of the integrin alpha10 on these stemcells by FACS analysis with the mAb365 antibody, approx 30% of the cellswere shown to be alpha10 positive. The results are shown in FIG. 12.

These results indicate a subpopulation of human mesenchymal stem cellsexpress the integrin alpha10.

Example 13 MAb 365 Stimulates Collagen Type II Production in MouseChondrocytes Objective

The objective with the present example is to study signalling functionof mAb365 via alpha10.

Materials and Methods

Mouse rib chondrocytes from C57 bl6 mouse were isolated using theprotocol described previously in example 11 and cultured for two days inDMEM/F12 supplemented with 10% FCS, PEST and ascorbic acid 50 μg/ml. Thecells were trypsinized and seeded into 15 ml tubes. Cells were spun down150×g for 5 min and treated in the following manner: Control (noantibody), mAb365 (10 μg/ml) and control antibody (IgG2akappa) (10μg/ml).

Cells with antibody treatments were placed in a cell incubator at 37°C., 5% CO₂, for 4 days to form pellet cultures. Cells were then treatedagain for 24 hrs with Control (no antibody), mAb365 (10 μg/ml) andcontrol antibody (IgG2akappa) (10 μg/ml).

RNA was extracted from all pellets using an RNeasy kit (Qiagen). cDNAsyntheses were made with Superscript™ II Rnase H⁻ Reverse Transcriptase(Invitrogen)

Quantitative PCR was performed in a Light Cycler (Roche) using FastStartDNA Master SYBR Green I with the following conditions: 10 mindenaturation 95° C., followed by 40 cycles with an annealing temperatureof 65° C. with specific primers for each transcript.

Results

FIG. 13 demonstrates that mAb365 antibody stimulates the expression ofcollagen type II mRNA. The control medium or control antibody(IgG2akappa) did not affect collagen mRNA synthesis.

Example 14 MAb365 Detects the Integrin Alpha10Beta1 in Human TissueSamples Objective

The objective with this example is to show that the mAb365 can be usedto detect the expression of the integrin alpha10beta1 in a tissuesample, for example, articular cartilage or atherosclerotic plaque.

Materials and Methods

In order to detect the presence of the integrin alpha10beta1 in tissuesamples, for example, articular cartilage or atherosclerotic plaque, thefollowing method is used:

-   -   1. Tissue is embedded in OCT, and 5 μm sections cut.    -   2. Sections are allowed to warm up to room temperature,        surrounded by PAP pen, and are then fixed in acetone for 10        minutes at −20° C.    -   3. Slides are washed in PBS for 15 minutes, and then incubated        with hyaluronidase (2 mg/ml in PBS; Sigma), at 37° C. for 30        minutes.    -   4. A second 15 minute wash in PBS is performed before incubating        sections with 2% donkey serum (Jackson, West Grove, Pa.) diluted        in PBS for 30 minutes to block. Sections are then incubated with        mAb365 diluted 1:400 in 2% donkey serum in PBS, for 60 minutes        at room temperature, and then washed in PBS for 15 minutes.    -   5. Incubation with the donkey-anti-mouseCy3 (Jackson) diluted in        PBS is performed for 60 minutes at room temperature, followed by        a final 15 minute wash in PBS. Slides can then be mounted with        Vectashield, and observed under a fluorescence microscope.

Results

Analysis of normal human articular cartilage by immunohistochemistryusing the mAb365 antibody shows that the integrin alpha10beta1 can bedetected in the tissue, in FIG. 14 represented by two different humanspecimens of 19 years and 53 years of age.

Example 15 Labelling the mAb365 with Biotin for Detection of theAntibody In Vivo Objective:

The objective with this example is to label the mAb365 with, forexample, biotin. Labelled antibody is then used in a method fordetecting the antibody in vitro and in vivo.

Materials and Methods

Biotinylation of mAb365: mAb365 is reacted with 4 μl 15 mg/ml biotin NHS(Vector Labs), plus 10 μg of mAb365 antibody in a total volume of 50 μlwith 100 mM Hepes (pH 8.5) for 2 hrs at room temp.

The sample is then dialysed in a volume of 500 ml for 30 mins with onechange of 100 mM Hepes. Dialysis is performed in a slide-a-lyzer minidialysis unit from Pierce (10,000 MWCO). The mAb365 is then collectedand ready for use.

Injection of Biotinylated mAb365

One-week-old C57 mice are injected intraperitoneally (25 G needle) with60 μl (=60 μg) mAb-BIOT (biotinylated mAb365) followed by a secondinjection after 6-8 hours with 50 μl (=50 μg) mAb-BIOT. Antibody titreis checked in serum by ELISA.

Back limbs are dissected out and embedded in OCT using standard methodssuch as Current Protocols in Molecular Biology. Vol 2 Ch 14. Ed: Ausubelet al (1991). Tissue is placed in a plastic mold with either the medialside down or plantar side down. The mold is then filled with OCT so thatthe tissue is completely covered, prior to placing on a copper plate ondry ice to freeze the tissue. Tissue blocks are stored at −20° C.Sections (5 μm) are cut on a cryostat, and slides are stored at −20° C.

Staining

Slides are allowed to come to room temperature for 30 minutes, and arethen fixed in acetone for 10 mins at −20° C. After washing in PBS for7+8 mins, sections are blocked in 2% donkey serum (diluted in PBS) for30 minutes at room temperature. The sections were then incubated withSA-Cy3 (diluted 1:200 in 2% donkey serum) for 60 mins at roomtemperature. The slides are washed 7+8 mins in PBS, and coverslipsmounted with Vectashield.

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1. A monoclonal antibody or a fragment thereof capable of binding specifically to the extracellular I-domain of the integrin alpha10 chain.
 2. A monoclonal antibody or a fragment thereof according to claim 1, wherein the antibody or fragment thereof is of murine origin.
 3. A monoclonal antibody or a fragment thereof according to claim 1, wherein the antibody or fragment thereof is humanized.
 4. A monoclonal antibody or a fragment thereof according to claim 1, wherein the fragment is selected from the group consisting of Fv, Fab, Fab′, F(ab′)₂ and single chain antibodies. 5-6. (canceled)
 7. A method for isolating a population of mammalian mesenchymal stem cells, the method comprising the steps of: a) providing a cell suspension comprising mammalian mesenchymal stem cells, b) contacting the cell suspension with a monoclonal antibody or a fragment thereof according to claim 1, under conditions wherein said monoclonal antibody or fragment thereof forms an antibody-antigen complex with the extracellular domain of integrin alpha10beta1, c) separating cells binding to the monoclonal antibody or fragment thereof in b), and optionally d) recovering cells binding to the monoclonal antibody or fragment thereof in c) from said antibody or fragment thereof, thereby producing a population of mammalian mesenchymal stem cells, optionally free from said antibody or fragment thereof.
 8. A method for isolating a population of mammalian chondrocytes, the method comprising the steps of: a) providing a cell suspension comprising chondrocytes, b) contacting the cell suspension with a monoclonal antibody or a fragment thereof according to claim 1, under conditions wherein said monoclonal antibody or fragment thereof forms an antibody-antigen complex with the extracellular I-domain of integrin alpha10beta1, c) separating cells binding to the monoclonal antibody or fragment thereof in b), and optionally d) recovering cells binding to the monoclonal antibody or fragment thereof in c) from said antibody or fragment thereof, thereby producing a population of chondrocytes, optionally free from said antibody or fragment thereof.
 9. A method for isolating a sub-population of mammalian ES cells, the method comprising the steps of: a) providing a cell suspension comprising ES cells, b) contacting the cell suspension with a monoclonal antibody or a fragment thereof according to claim 1, under conditions wherein said monoclonal antibody or fragment thereof forms an antibody-antigen complex with the extracellular I-domain of integrin alpha10beta1, c) separating cells binding to the monoclonal antibody or fragment thereof in b), and optionally d) recovering cells binding to the monoclonal antibody or fragment thereof in c) from said antibody or fragment thereof, thereby producing a sub-population of mammalian ES cells, optionally free from said antibody or fragment thereof.
 10. A method according to claim 7, wherein the monoclonal antibody or fragment thereof is linked to a solid phase.
 11. A method according to claim 7, wherein the solid phase is beads.
 12. A method according to claim 7, wherein the mammalian cells are human cells.
 13. A method according to claim 7, wherein the mammalian cells are murine cells.
 14. A population of mammalian mesenchymal stem cells, wherein the population is obtained by a method according to claim
 7. 15. A population of mammalian mesenchymal stem cells according to claim 14, wherein the stem cells are human mesenchymal stem cells.
 16. A population of mammalian mesenchymal stem cells according to claim 14, wherein the stem cells are murine mesenchymal stem cells.
 17. A population of mammalian chondrocytes, wherein the population is obtained by a method according to claim
 8. 18. A population of mammalian chondrocytes according to claim 17, wherein the chondrocytes are human chondrocytes.
 19. A population of mammalian chondrocytes according to claim 17, wherein the chondrocytes are murine chondrocytes.
 20. A subpopulation of mammalian ES cells, wherein the subpopulation is obtained by a method according to claim
 9. 21. A subpopulation of mammalian ES cells according to claim 20, wherein the ES cells are human ES cells.
 22. A subpopulation of mammalian ES cells according to claim 20, wherein the ES cells are murine ES cells.
 23. A method for detecting a mesenchymal stem cell in a sample, the method comprising the steps of: a) providing a sample cell suspension comprising a mesenchymal stem cell, b) contacting said sample cell suspension with a monoclonal antibody or a fragment thereof according to claim 1, c) incubating the sample cell suspension and the monoclonal antibody or fragment thereof under conditions wherein said monoclonal antibody or fragment thereof forms an antibody-antigen complex with the extracellular domain of integrin alpha10beta1 on a mesenchymal stem cell, d) optionally adding a second labeled antibody or a fragment thereof to the sample cell suspension, wherein the second antibody or fragment thereof binds to the monoclonal antibody or fragment thereof of c), and e) detecting the monoclonal antibody or fragment thereof of c), or optionally detecting the second labeled antibody or fragment thereof of d), thereby detecting the mesenchymal stem cell.
 24. A method for detecting a chondrocyte in a sample, the method comprising the steps of: a) providing a sample cell suspension comprising a chondrocyte, b) contacting said sample cell suspension with a monoclonal antibody or a fragment thereof according to claim 1, c) incubating the sample cell suspension and the monoclonal antibody or fragment thereof under conditions wherein said monoclonal antibody or fragment thereof forms an antibody-antigen complex with the extracellular domain of integrin alpha10beta1 on a chondrocyte, d) optionally adding a second labeled antibody or a fragment thereof to the sample cell suspension, wherein the second antibody or fragment thereof binds to the monoclonal antibody or fragment thereof of c), and e) detecting the monoclonal antibody or fragment thereof of c), or optionally detecting the second labeled antibody or fragment thereof of d), thereby detecting the chondrocyte.
 25. A method for detecting an ES cell in a sample, the method comprising the steps of: a) providing a sample cell suspension comprising an ES cell, b) contacting said sample cell suspension with a monoclonal antibody or a fragment thereof according to claim 1, c) incubating the sample cell suspension and the monoclonal antibody or fragment thereof under conditions wherein said monoclonal antibody or fragment thereof forms an antibody-antigen complex with the extracellular domain of integrin alpha10beta1 on an ES cell, d) optionally adding a second labeled antibody or a fragment thereof to the sample cell suspension, wherein the second antibody or fragment thereof binds to the monoclonal antibody or fragment thereof of c), and e) detecting the monoclonal antibody or fragment thereof of c), or optionally detecting the second labeled antibody or fragment thereof of d), thereby detecting the ES cell.
 26. A method for blocking the binding of a chondrocyte to an extracellular matrix (ECM) molecule, the method comprising the steps of: a) providing a chondrocyte and an ECM molecule, b) contacting the chondrocyte with a monoclonal antibody or a fragment thereof according to claim 1, under conditions wherein said monoclonal antibody or fragment thereof forms an antibody-antigen complex with the extracellular domain of integrin alpha10beta1, and c) incubating said antibody-antigen complex, thereby blocking the binding of the chondrocyte to the ECM molecule.
 27. A method for modulating the signaling of alpha10beta1 in a mammalian mesenchymal stem cell, ES cell or chondrocyte, the method comprising the steps of: a) providing a mammalian mesenchymal stem cell, ES cell or chondrocyte, b) contacting the mesenchymal stem cell, ES cell or chondrocyte with a monoclonal antibody or a fragment thereof according to claim 1, under conditions wherein said monoclonal antibody or fragment thereof forms an antibody-antigen complex with the extracellular domain of integrin alpha10beta1 on said cells, and c) incubating said antibody-antigen complex, thereby modulating the signaling of alpha10beta1 in the mesenchymal stem cell, ES cell or chondrocyte.
 28. A method for detecting the expression of integrin alpha10beta1 in a tissue sample or on a cell surface, the method comprising the steps of: a) providing a tissue sample or a cell sample, b) contacting the sample with a monoclonal antibody or a fragment thereof according to claim 1, c) incubating the sample and the monoclonal antibody or fragment thereof under conditions wherein said monoclonal antibody or fragment thereof forms an antibody-antigen complex with the extracellular domain of integrin alpha10beta1, d) optionally adding a second labeled antibody or a fragment thereof to the sample, wherein the second antibody or fragment thereof binds to the monoclonal antibody or fragment thereof of c), and e) detecting the monoclonal antibody or fragment thereof of c), or optionally detecting the second labeled antibody or fragment thereof of d).
 29. A method for in vivo imaging the expression of the integrin alpha10beta1 in a mammal, the method comprising the steps of: a) providing a mammal, b) providing a monoclonal antibody or a fragment thereof according to claim 1, wherein said monoclonal antibody or a fragment thereof optionally are conjugated, c) administering the monoclonal antibody or fragment thereof to the mammal, under conditions wherein the antibody or fragment thereof binds to the extracellular I-domain of integrin alpha10beta1 of cells in said mammal, d) optionally administering a second labeled antibody or a fragment thereof to the mammal, wherein the second antibody or fragment thereof binds to the monoclonal antibody or fragment thereof of c), e) detecting the monoclonal antibody or fragment thereof of c), or optionally detecting the second labeled antibody or fragment thereof of d), and f) creating an image of the detected antibody or fragment thereof, thereby in vivo imaging the expression of integrin alpha10beta1 on cells in a mammal.
 30. A method according to claim 29, wherein the extracellular I-domain of integrin alpha10beta1 is on a cell in an atherosclerotic plaque in a blood vessel.
 31. A composition comprising a monoclonal antibody or a fragment thereof according to claim
 1. 32. A composition according to claim 31 wherein the monoclonal antibody or fragment thereof further comprises a detectable label.
 33. An administration vehicle comprising a monoclonal antibody or a fragment thereof according to claim
 1. 34. An administration vehicle comprising a monoclonal antibody or a fragment thereof according to claim 1, a pharmaceutically acceptable carrier, and a pharmaceutically acceptable drug affecting joint diseases or atherosclerosis.
 35. A pharmaceutical composition for the treatment of musculoskeletal diseases, arthritis or atherosclerosis comprising a monoclonal antibody or a fragment thereof according to claim
 1. 36. A pharmaceutical composition for gene therapy treatment of musculoskeletal diseases, arthritis or atherosclerosis comprising a monoclonal antibody or a fragment thereof according to claim 1, wherein the monoclonal antibody or fragment thereof targets gene delivery to integrin alpha10 beta1 expressing cells.
 37. A pharmaceutical composition according to claim 36 comprising an adenovirus for gene therapy treatment of arthritis.
 38. A kit comprising a monoclonal antibody or a fragment thereof according to claim
 1. 39. A kit according to claim 38, wherein the monoclonal antibody or fragment thereof is bound to a solid phase.
 40. A kit according to claim 38, wherein the monoclonal antibody or fragment thereof comprises a detectable label.
 41. (canceled)
 42. A method for making a monoclonal antibody or a fragment thereof according to claim 1, the method comprising the steps of: a) immunizing and boosting an alpha-10 knock-out mouse with recombinant alpha-10 I-domain; b) fusing the spleen cells from the immunized mouse with immortalized cells to create hybridoma cells; and c) culturing the hybridoma cells and isolating the antibodies produced thereby.
 43. A method according to claim 42 wherein the immortalized cells are NSO cells.
 44. A monoclonal antibody or a fragment thereof produced by a method according to claim
 42. 45. A monoclonal antibody or a fragment thereof produced by a method according to claim 42, wherein the antibody is not produced by the hybridoma cell line deposited at the Deutsche Sammlung von Microorganismen und Zellkulturen GmbH under the accession number DSM ACC2583.
 46. A monoclonal antibody or a fragment thereof according to claim 45, wherein the antibody or fragment thereof is humanized.
 47. A monoclonal antibody or a fragment thereof according to claim 45, wherein the fragment is selected from the group consisting of Fv, Fab, Fab′, F(ab′)₂ and single chain antibodies. 